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The role of Stp1, a secreted effector, in the biotrophic interaction of
Ustilago maydis and its host plant maize
Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften (Dr. rer. nat.)
Dem Fachbereich Biologie der Philipps-Universität Marburg
vorgelegt von
Liang Liang aus Hebei/P. R. China
Marburg/Lahn, 2012
Die Untersuchungen zur vorliegenden Arbeit wurden von Anfang January 2009 bis June
2012 unter der Betreuung von Frau Prof. Dr. Regine Kahmann in Marburg am Max-Planck-
Institut für terrestrische Mikrobiologie in der Abteilung Organismische Interaktionen
durchgeführt.
Vom Fachbereich Biologie
der Philipps-Universität Marburg als Dissertation
angenommen am: 28.12.2012
Erstgutachter: Frau Prof. Dr. Regine Kahmann
Zweitgutachter: Herr Prof. Dr. Michael Bölker
Tag der mündlichen Prüfung: 08.02.2013
Declaration I hereby declare that the dissertation entitled “The role of Stp1, a secreted effector, in the
biotrophic interaction of Ustilago maydis and its host plant maize” submitted to the
Department of Biology, Philipps-Universität Marburg, is the original and independent work
carried out by me under the guidance of the PhD committee, and the dissertation is not
formed previously on the basis of any award of Degree, Diploma or other similar titles.
- Marburg, Oct 2012
Liang Liang
Contents Abbreviations ........................................................................................................................ I
Summary .............................................................................................................................. II
Zusammenfassung ............................................................................................................. III
1. Introduction .............................................................................................................. 1
1.1 The Ustilago maydis/Zea mays pathosystem ......................................................................... 1
1.1.1 Maize, an economic important model organism for fundamental research .......................................... 1
1.1.2 Ustilago maydis as a model organism .................................................................................................. 1
1.1.3 Life cycle of U. maydis ........................................................................................................................ 2
1.2 Plant defense response and R proteins ................................................................................... 3
1.2.1 PAMP-triggered immunity ................................................................................................................... 4
1.2.2 Effector-triggered immunity ................................................................................................................ 4
1.2.3 Systemic acquired resistance ................................................................................................................ 5
1.3 Secreted effectors in host-pathogen interactions .................................................................... 5
1.3.1 Translocation and function of bacteria effectors .................................................................................. 6
1.3.2 Translocation and function of oomycetes effectors .............................................................................. 7
1.3.3 Identification and function of fungal effectors ..................................................................................... 8
1.4 Co-evolution of plants and their microbial pathogens ........................................................... 9
1.5 Stp1 plays crucial role in the establishment of biotrophic interaction between U. maydis and
maize ……………………………………………………………………………………………...10
1.6 Aims of this study ................................................................................................................ 12
2. Results ...................................................................................................................... 13
2.1 Functional domain analysis of Stp1 ..................................................................................... 13
2.1.1 The variable domains of Stp1 are dispensable ................................................................................... 13
2.1.2 N- and C-termini of Stp1 could be separately expressed ................................................................... 17
2.1.3 Stp1 related proteins in other smut fungi can replace Stp1 of U. maydis ........................................... 19
2.1.4 The putative functional domains show low similarity with known functional domains in the
databases are not valid ...................................................................................................................................... 20
2.2 Interactors of Stp1 and Stp1∆136-432 are both cytoplasmic and apoplastic maize proteins .... 22
2.2.1 Interactors identified by full-length Stp1 are not likely to be functionally relevant ........................... 22
2.2.2 Interactors of Stp1∆136-432 are both cytoplasmic and apoplastic maize proteins .................................. 23
2.2.3 N- and C-termini of Stp1 may play separate functions ...................................................................... 26
2.2.4 Stp1 can interact with several cysteine proteases of maize ................................................................ 28
2.2.5 Comparison of the interactors with microarray data .......................................................................... 29
2.3 Purification of recombinant Stp1 protein ............................................................................. 29
2.3.1 Purification of His-Stp1∆136-432 ........................................................................................................... 29
2.3.2 Purification of His-Stp129-135, His-Stp1433-515 and His-Aro7 ............................................................... 32
2.3.3 Purification of Strep-Sip3. ................................................................................................................. 34
2.4 The C-terminus of Stp1 inhibits the activity of the maize cysteine protease, Sip3 .............. 34
2.5 Localization of Stp1 in infected plants ................................................................................. 35
2.6 Differential expression analysis of U. maydis infected plants by RNA-Seq ........................ 37
2.6.1 Sequencing and mapping of reads to the maize genome .................................................................... 37
2.6.2 Strategy for detection of differentially expressed genes .................................................................... 39
2.6.3 SG200∆stp1-stp1∆40-136 triggered distinct plant responses from SG200∆stp1-stp1∆432-515 .................. 40
3. Discussion ................................................................................................................ 54
3.1 Domain structure of Stp1 ..................................................................................................... 54
3.1.1 The N- and C-terminal conserved domains of Stp1 are essential for protein function while the
variable domains are dispensable. .................................................................................................................... 54
3.1.2 N- and C-terminal domains of Stp1 may be essential for the stability of each other ......................... 55
3.2 The interaction partners of Stp1 ........................................................................................... 55
3.2.1 The biological significance of the inhibition of Sip3 by the C-terminus of Stp1 ............................... 56
3.2.2 The cytoplasmic maize interaction partners of Stp1 shed light on a putative function of Stp1 in the
plant cytosol ..................................................................................................................................................... 57
3.3 Stp1, an effector with apoplastic and cytoplasmic functions? ............................................. 59
3.4 Glycine-rich domain of Stp1 may promote fungal growth in vascular bundles. .................. 60
3.5 The N- and C-terminal domains of Stp1 appear to have distinct functions ......................... 61
3.5.1 Several early defense response genes were not induced by stp1 mutants expressing the N-terminus of
Stp1 ………………………………………………………………………………………………………..62
3.5.2 stp1 mutants expressing the C-terminus of Stp1 triggered stronger plant defense response than stp1
mutants ………………………………………………………………………………………………………..63
3.6 Working model of the function of Stp1 ............................................................................... 64
4. Materials and methods ........................................................................................... 66
4.1 Materials and source of supplies .......................................................................................... 66
4.1.1 Chemicals and enzymes ..................................................................................................................... 66
4.1.2 Buffers and solutions .......................................................................................................................... 66
4.1.3 Kits ..................................................................................................................................................... 66
4.2 Media ................................................................................................................................... 67
4.2.1 Media for E. coli and A. tumefaciens ................................................................................................. 67
4.2.2 Media for U. maydis ........................................................................................................................... 67
4.2.3 Media for S. cerevisiae ....................................................................................................................... 68
4.3 Strains ................................................................................................................................... 69
4.3.1 Escherichia coli strains ...................................................................................................................... 69
4.3.2 Agrobacterium tumefaciens strain ...................................................................................................... 69
4.3.3 Ustilago maydis strains ...................................................................................................................... 69
4.3.4 Saccharomyces cerevisiae strains ...................................................................................................... 71
4.4 Oligonucleotides .................................................................................................................. 71
4.5 Plasmids ............................................................................................................................... 73
4.5.1 Plasmids for generation of U. maydis mutants ................................................................................... 73
4.5.2 Plasmids for Y2H assays .................................................................................................................... 74
4.5.3 Plasmids for protein expression ......................................................................................................... 76
4.6 Microbiological methods ..................................................................................................... 77
4.6.1 E. coli and A. tumefaciens methods .................................................................................................... 77
4.6.2 U. maydis methods ............................................................................................................................. 79
4.6.3 S. cerevisiae methods ......................................................................................................................... 80
4.7 Molecular biological methods .............................................................................................. 82
4.7.1 Southern blotting ................................................................................................................................ 82
4.7.2 Western blotting ................................................................................................................................. 83
4.7.3 Isolation of Plasmid DNA from S. cerevisiae .................................................................................... 84
4.7.4 Protein extraction from S. cerevisiae ................................................................................................. 84
4.8 Biochemical methods ........................................................................................................... 84
4.8.1 Purification of GST-tagged protein .................................................................................................... 85
4.8.2 Purification of Strep-tagged protein ................................................................................................... 85
4.8.3 Purification of His-tagged protein ...................................................................................................... 86
4.8.4 Protein purification from N. benthamiana ......................................................................................... 87
4.8.5 Cysteine pretease activity and inhibition assay .................................................................................. 89
4.9 Staining and microscopy observation .................................................................................. 89
4.9.1 WGA-AF488 / Propidium Iodide staining ......................................................................................... 89
4.9.2 Anniline blue / Propidium Iodide staining ......................................................................................... 90
4.9.3 Chlorazol Black E staining ................................................................................................................. 90
5. References ............................................................................................................... 91
Supplementary data……………………………………………………………………....99
Acknowledgements ........................................................................................................... 100
Curriculum Vitae .............................................................................................................. 101
I
Abbreviations
Δ Deletion LC-MS Liquid chromatography mass spectrometry
A Adenine Leu L-Leucine
aa amino acid M Molar
AD activiation domain mA milliampere
Ade L-Adenine hemi-sulfate salt min Minute
Amp Ampicillin ml milliliter
BD DNA binding domain mM Millimolar
CBX Carboxin NB nucleotide binding
cDNA complementary DNA NLS nuclear localization signal
CP cysteine protease N-terminal amino-terminal
C-terminal Carboxyl-terminal N-terminus amino-terminus
C-terminus Carboxyl-terminus OD600 Optical density at 600 nm
CV colume volume P/MAMPs Pathogen/microbe associated molecular patterns
DAPI 4',6-diamidino-2-phenylindole
PEG Polyethylene glycol
dH2O distilled water PI Propidium Iodide
DMSO Dimethyl sulphoxide PPRs pattern recognition receptors
dpi days post infection PTI PAMP-triggered immunity
EDTA Ethylene Diamine Tetraacetic Acid
Rif Rifampicin
ETI effector-triggered immunity rpm revolutions per minute
GO gene ontology RT-PCR Real time PCR or reverse transcription PCR
GST glutatione S-transferase S second
h hour SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
HA Hemagglutinin Sip Stp1 interacting protein
His Histidine/L-Histidine HCl monohydrate
stp1 stop after penetration
hpi hours post infection Try L-Tryptophan
HR hypersensitive response V Voltage
IP Immuno-precipitation w/v weight/volume
IPTG isopropyl β-D-1-thiogalactopyranoside
WGA wheat germ agglutinin
Kan Kanamycin Y2H yeast two-hybrid
kDa kilodalton μl microliter
II
Summary
Secreted effectors play crucial roles during the establishment of the biotrophic interaction
between Ustilago maydis and maize. In a previous study (Schipper, 2009) it had been
demonstrated that a deletion of the stp1 effector gene resulted in a complete loss of
virulence symptoms in maize infection and that such mutants elicited a hypersensitive
response. This distinguishes stp1 from most other secreted effectors that are either
dispensable for pathogenicity or have only a minor effect on virulence. This study focuses
on the functional analysis of Stp1.
A mutational analysis showed that the conserved N- and C-terminal domains of Stp1 can be
separately expressed but are both required for Stp1 protein function. The long central
variable domain was demonstrated to be dispensable yet may promote fungal growth in
vascular bundles. stp1 homologs from closely related smut fungi of U. maydis could
replace stp1 in U. maydis, indicating a conserved function. Stp1∆136-432 lacking the central
domain could be purified to homogeneity and was stable, while the isolated C-terminal
domain, Stp1433-515, was unstable after purification. This could suggest that N- and C-
terminal domains of Stp1 stabilize each other. Stp1-HA expressed by U. maydis was
detected in the nucleus of plant cells by immunolocalization suggesting that Stp1 may
suppress plant defense responses by affecting the transcription of respective genes.
Both cytoplasmic and apoplastic maize proteins were identified as interaction partners of
Stp1 by yeast two-hybrid assays using Stp1∆136-432 as bait, suggesting that Stp1 may be an
effector with both apoplastic and cytoplasmic functions The C-terminus of Stp1 as well as
Stp1∆136-432 could inhibit the activity of a maize extracellular cysteine protease, Sip3, which
was identified as one of the apoplastic interaction partners. The interactions between Stp1
and the cytoplasmic interactors Sip9, a cell number regulator 8, Sip16, a CCR4-NOT
transcription complex subunit, Sip19, a serine/threonine-protein kinase and Sip21, a VIP2
protein were verified with full-length cDNA but await to be confirmed by other techniques.
RNA-Seq analysis demonstrated that several early defense response genes are not induced
by stp1 mutants expressing the N-terminus of Stp1 while stp1 mutants expressing the C-
terminus of Stp1 triggered even stronger plant defense responses than stp1 mutants during
colonization. This suggests that N- and C-terminal domains of Stp1 have distinct functions.
III
Zusammenfassung
Bei der Etablierung der biotrophen Interaktion zwischen Ustilago maydis und Mais spielen
sekretierte Effektoren eine entscheidende Rolle. In einer vorausgegangenen Studie
(Schipper, 2009) konnte gezeigt werden, dass die Deletion des stp1 Effektor-Gens zu einem
vollständigen Verlust der Virulenz führt und dass entsprechende Mutanten eine
hypersensitive Reaktion auslösen. Diesbezüglich unterscheidet sich stp1 von den meisten
anderen untersuchten Effektoren, die entweder keinen oder nur einen geringen Beitrag zur
Virulenz des Pilzes leisten. Diese vorliegende Arbeit befasst sich mit der funktionellen
Analyse von Stp1.
Mutationsanalysen zeigten, dass die konservierten N- und C-terminalen Domänen von Stp1
zwar als separate Polypeptide exprimiert werden können, aber die Gegenwart beider
Domänen nötig ist, um die Stp1 Funktion zu komplementieren. Es wurde nachgewiesen,
dass die zentrale variable Domäne von Stp1 keine essentielle Funktion übernimmt,
allerdings könnte diese Domäne für die Proliferation des Pilzes entlang der Leitbündel eine
Rolle spielen. stp1 Homologe aus mit U. maydis nahe verwandten Brandpilzen waren in der
Lage, die Funktion von stp1 in U. maydis zu komplementieren, was für eine konservierte
Funktion spricht. Eine Stp1 Version, bei der die zentrale Domäne deletiert wurde (Stp1∆136-
432), konnte bis zur Homogenität aufgereinigt werden und erwiess sich als stabil, während
die isolierte C-terminale Domäne, Stp1433-515, nach der Aufreinigung instabil war. Diese
Beobachtung könnte darauf hinweisen, dass sich die N- und C-terminalen Domänen von
Stp1 gegenseitig stabilisieren. Durch Immunlokalisierung konnte ein Stp1-HA
Fusionsprotein im Zellkern von Pflanzenzellen detektiert werden, die mit dem Stamm
SG200stp1-HA infiziert waren. Demzufolge könnte Stp1 auf die Transkription von Genen
Einfluss nehmen und dadurch die pflanzliche Abwehrreaktion unterdrücken.
In einem Hefe-zwei-Hybrid-System, bei dem Stp1 als Köder benutzt wurde, konnten
sowohl apoplastische als auch cytoplasmatische Mais Proteine als Stp1-Interaktoren
identifiziert werden. Dies könnte bedeuten, dass Stp1 ein Effektorprotein mit dualer
Funktion zum einen im Apoplasten und zum anderen im pflanzlichen Zytoplasma ist.
Sowohl die gereinigte C-terminale als auch die N-terminale Domäne von Stp1war in der
Lage, die Aktivität der extrazellulären Mais Cystein-Protease Sip3 zu inhibieren, die zuvor
IV
als ein apoplastischer Interaktionspartner von Stp1 identifiziert werden konnte. Die
Interaktion zwischen Stp1 und den cytoplasmatischen Interaktoren Sip9 (cell number
regulator 8), Sip16(CCR4-NOT Transkriptionskomplex Untereinheit), Sip19
(Serine/Threonin- Kinase) und demVIP2 Protein Sip21 konnten nach Expression der
jeweiligen Gene in voller Länge verifiziert werden, müssen aber zukünftig noch durch
weitere Methoden bestätigt werden. RNA-Seq Analysen des Transkriptoms infizierter Mais
Blätter zeigten, dass einige der frühen pflanzlichen Abwehrgene, die nach Infektion mit
einem stp1-Deletions Stamm induziert werden, Hingegen führte die Infektion mit einem
stp1-Deletions Stamm komplementiert durch den Stp1 C-Terminus sogar zu einer stärkeren
Abwehrreaktion als sie nach Infektion mit dem stp1- Deletionsstamm beobachtet wurde.
Dies weist darauf hin, dass die N- und C-terminale Domänen von Stp1 unterschiedliche
Funktionen erfüllen könnten.
Introduction
1
1. Introduction
1.1 The Ustilago maydis/Zea mays pathosystem
The Ustilago maydis–maize pathosystem has emerged as the current model for plant
pathogenic basidiomycetes and as one of the models for biotrophic interaction (Brefort et
al., 2009).
1.1.1 Maize, an economic important model organism for fundamental research
Maize (Zea mays), the domesticated variant of mesoamerican teosinte is subjected to
cultivation and selection since 4,200 years B.C. (Benz, 2001). In 2010, 844 million tonnes
of maize was produced worldwide, more than rice (672 million tonnes) and wheat (651
million tonnes) (Food and Agriculture Organization of the United Nations, 2010). Beyond
its major agricultural and economic contributions, maize is an important model organism
for fundamental research in the inheritance and functions of genes, the physical linkage of
genes to chromosomes, the mechanistic relation between cytological crossovers and
recombination, the origin of the nucleolus, the properties of telomeres, epigenetic silencing,
imprinting, and transposition (Schnable et al., 2009, Bennetzen, 2009). An improved draft
nucleotide sequence of the 2.3 gigabase genome of maize is published in 2009 and ever
since it is undergoing continuous update (Schnable et al., 2009). In the latest release,
39,656 genes (63,540 transcripts) in the filtered gene set are annotated
(http://www.maizesequence.org) which will significantly promote fundamental research on
maize and related grasses.
1.1.2 Ustilago maydis as a model organism
All aerial parts of maize can be infected by the facultatively biotrophic fungus, Ustilago
maydis. The infection leads to disease symptoms like chlorosis, ligular swellings and
tumors (Kamper et al., 2006). U. maydis, which is pathogenic only on corn and its close
relative teosinte, belongs to the basidiomycetes, a group of fungi that includes the common
mushroom and many plant pathogens such as the smuts and rusts (Banuett, 1992). Several
features make this microorganism a model for a number of important cellular processes
such as signalling, dimorphism, DNA recombination and repair, plant microbe interactions
etc (Bolker, 2001, Kamper et al., 2006, Holliday, 2004). U. maydis can be grown in defined
media. The fungus is haploid, grows by budding and forms compact colonies on plates that
Introduction
2
can be replica plated (Dean et al., 2012). U. maydis can induce prominent disease
symptoms (tumors) on all aerial parts of maize within less than a week (Brefort et al.,
2009). In addition, solopathogenic, haploid strains are instrumental for the study of
pathogenesis (Bölker, 1995). A number of molecular tools are available such as a PCR
based gene replacement strategy, inducible promoter systems and fluorescent protein based
localization techniques (Kamper, 2004, Basse & Steinberg, 2004, Doehlemann et al.,
2009). One of the major breakthroughs for the U. maydis research community was the
public release of the genome sequence combined with a solid manual annotation that
resulted in high-quality data currently curated in the database MUMDB at MIPS
(Vollmeister et al., 2012, Kamper et al., 2006).
1.1.3 Life cycle of U. maydis
The dimorphic fungus, U. maydis, exhibits three distinct morphological forms in its life
cycle. Haploid cells, which are nonpathogenic, are saprophytic and grow in a yeast-like
unicellular form (sporidium) that divides by budding (Fig. 1A) (Perez-Martin et al., 2006).
Fusion of two compatible haploid cells, which harbor different alleles of a and b mating
types loci, is required to generate the filamentous dikaryon which is strictly dependent on
the host plant for sustained growth (Fig. 1B) (Bolker, 2001, Kahmann, 2000). The
dikaryotic hyphae show tip-directed growth and cytoplasm accumulates in the tip cell
compartment, whereas, older parts of the hyphae become vacuolated and are sealed off by
regularly spaced septa (Brefort et al., 2009). The pathogenic dikaryotic differentiating
appresoria are able to penetrate plant cells (Fig. 1C). During penetration, the host plasma
membrane invaginates and tightly surrounds the intracellular hyphae (Brefort et al., 2009).
An interaction zone develops between plant and fungal membranes that is characterized by
fungal deposits produced by exocytosis (Kamper et al., 2006). After penetration, the hyphae
first proliferate intracellularly (Fig. 1D) and then at later stages accumulate in mesophyll
tissue and are found mostly in apoplastic cavities that arise in the developing tumors
(Doehlemann et al., 2009). Massive proliferation is followed by nuclear fusion and
fragmentation of the hyphae, a process that releases individual cells that will produce the
diploid spore (teliospore, Fig. 1 E and F) (Perez-Martin et al., 2006). Fungal proliferation is
associated with development of the prominent disease symptoms of U. maydis, tumors (Fig.
1 G).
Introduction
3
1.2 Plant defense response and R proteins
Although plants are under continuous attack by pathogens, most encounters result in plant
resistance and disease being the exception (Takken & Joosten, 2000). Passive protection
against pathogens that are not specialized to attack is provided by cell walls, wax layers and
preformed chemical barriers (Jones & Dangl, 2006). In case a pathogen overcomes these
obstacles, plants have evolved three different strategies of defense namely, PAMP-triggered
immunity, effector-triggered immunity and systematic acquired resistance.
Fig.1. Life cycle of U. maydis. A: Scanning electron microscopy (SEM) image of haploid sporidia. B: SEM image of mated sporidia on plant epidermis; arrow denotes dikaryotic filament. C: Confocal image of hyphae after penetration stained with WGA-AF488 and propidium iodide (WGA/PI), the arrow denotes appresorium. D: Confocal image of hyphae proliferating on planta stained with WGA/PI. E: SEM image of sporogenous hyphae and early stages of spore development. F: SEM image of ornamented teliospores. G, Tumors formed on the ear of maize. Scale bars in A, B E and F, 5 µm. Scale bars in C and D, 20 µM. Figure A, B E and F were modified from Kamper et al. (2006).
Introduction
4
1.2.1 PAMP-triggered immunity
Pathogen/microbe associated molecular patterns (PAMPs, also called MAMPs) are
recognized by receptor-like proteins or kinases (RLP/Ks) termed pattern recognition
receptors (PRRs) (Dodds & Rathjen, 2010). PAMPs are typically essential components of
whole classes of pathogens, such as bacterial flagellin or fungal chitin. Plants also respond
to endogenous molecules released by pathogen invasion, such as cell wall or cuticular
fragments called danger-associated molecular patterns (DAMPs). Stimulation of PRRs
leads to PAMP-triggered immunity (PTI) including rapid ion fluxes across the plasma
membrane, MAP kinase activation, production of reactive-oxygen species, rapid changes in
gene expression and cell wall reinforcement (Zipfel, 2008). Successful pathogens have
evolved strategies to infect host plants, either by evading recognition or by suppressing the
subsequent signalling steps. In many cases, suppression of PTI involves secretion of
virulence molecules by the pathogens called effectors (Jones & Dangl, 2006).
1.2.2 Effector-triggered immunity
The second strategy of the perception of a pathogen is based on resistance (R) genes in
plants whose products confer recognition of cognate secreted virulence determinants from
the pathogen referred to as ‘effectors’ (Avr proteins). This recognition induces effector-
triggered immunity (ETI) which often culminates in a hypersensitive response (HR). This
gene-for-gene hypothesis was introduced by Flor in the 1940s, and dozens of R-Avr gene
combinations have since been characterized (Jones & Dangl, 2006, van der Hoorn &
Kamoun, 2008). Recognition events during ETI are mostly mediated by a class of receptor
proteins that contain nucleotide binding (NB) domains and leucine-rich repeat (LRR). NB-
LRR proteins can recognize pathogen effectors either directly by physical association or
indirectly through and accessory protein that is a pathogen virulence target or a structure
mimic of one (Dodds & Rathjen, 2010). Three conceptual models have been proposed to
describe the mechanism of indirect recognition. The ‘guard’ model postulates that NB-LRR
proteins guard an accessory protein (or guardee) that is targeted and modified by pathogen
effectors (Jones & Dangl, 2006). The ‘decoy’ model proposes that duplication of the
effector target gene or independent evolution of a target mimic could relax evolutionary
constraints and allow the accessory protein to participate solely in effector perception (van
der Hoorn & Kamoun, 2008). The ‘bait-and-switch’ model envisages a two-step
Introduction
5
recognition event. First, an effector interacts with the accessory ‘bait’ protein associated
with an NB-LRR, and then a subsequent recognition event occurs between the effector and
NB-LRR protein to trigger signalling (Collier & Moffett, 2009).
1.2.3 Systemic acquired resistance
Plants are also protected by a mechanism called systemic acquired resistance (SAR), which
occurs at sites distant from primary and secondary immune responses and protects plants
from subsequent pathogen attacks (de Wit, 2007). SAR is effective against a broad range of
pathogens and is dependent on different plant hormones including salicylic acid (SA),
jasmonic acid (JA), ethylene (ET), abscisic acid (ABA) or combinations thereof. SA and
SA derivatives are important for resistance to biotrophic pathogens that require living plant
cells for reproduction, while JA and JA conjugates cooperate with ET to regulate resistance
to necrotrophic pathogens that kill plant cells as they reproduce (Panstruga et al., 2009).
The SA and JA defense pathways are mutually antagonistic, and bacterial pathogens have
evolved to exploit this fact to overcome SA-mediated defense responses (Kunkel & Brooks,
2002).
1.3 Secreted effectors in host-pathogen interactions
The deployment of secreted effectors is postulated to be the key to host infection (Rafiqi et
al., 2012). Research on effectors secreted by pathogens including bacteria, oomycetes and
fungi during host invision has dominated the field of molecular plant–microbe interactions
over recent years (de Jonge et al., 2011). In a successful infection, pathogen effectors
facilitate suppression of the plant immune system and orchestrate the reprogramming of the
infected tissue so that it becomes a source of nutrients that are required by the pathogen to
support its growth and development (Koeck et al., 2011). Identification of bacterial
effector proteins has provided unparalleled insights into the evolution of bacterial
pathogenesis and host mimicry employed by bacterial proteins to interfere with host
signaling and signal transduction processes (Whisson et al., 2007). Important progress in
the study of oomycete effectors has been made leading to the identification of large
repertoires of effectors with characteristic RXLR and other motifs required for host cell
uptake, elucidation of the 3D structures of RXLR effectors, novel insights into how
Introduction
6
cytoplasmic effectors subvert host cells etc. (Bozkurt et al., 2012). However, the functions
of fungal effector proteins are only beginning to be revealed.
1.3.1 Translocation and function of bacteria effectors
Plant pathogenic bacteria require a type III secretion system (TTSS) to translocate effector
proteins into host cells to promote disease by altering the normal physiology of the plant in
favour of the pathogen (Abramovitch & Martin, 2005). The type III secretion system in
pathogenic bacteria consisits of 15–20 Hrp (hypersensitive response and pathogenicity)
proteins building a secretion apparatus that is involved in the secretion and translocation of
effector proteins to the plant cell (Cornelis & Van Gijsegem, 2000). The cloning of the first
bacterial avirulence gene, avrA, from Pseudomonas syringae pv. glycinea marked the
beginning of the molecular analysis of bacterial effectors and has paved the way for
determining of the role of bacterial effectors in pathogen virulence and the triggering of
plant innate immunity (Staskawicz, 2009).
Key modules of PAMP-triggered immunity signaling pathways are frequently targeted by
type III effectors (Feng & Zhou, 2012). The kinase domain of FLS2, EFR, and CERK1
constitutively interacts with BIK1, constituting a preformed immune receptor complex. The
perception of flg22 and elf18 by FLS2 and EFR recruits another receptor- like kinase called
BAK1 to activate the immune receptor complexes, leading to the phosphorylation of BIK1
and activation of downstream signaling. AvrPto and AvrPtoB directly target Arabidopsis
and tomato PAMP receptors FLS2, EFR, and CERK1 to block PTI. In addition, MAPK
cascades are similarly targeted by multiple P. syringae effectors such as HopAI1 and
HopF2 which inactivate MPKs and MKKs respectively. A significant portion of type III
effectors act by eliminating their host target proteins (Feng & Zhou, 2012). For example,
AvrPphB and AvrRpt2, the cysteine proteases, recognize and cleave BIK1 and RIN4
respectively. HopZ1 and HopM1 induce the degradation of their target proteins, GmHID1
and MIN7 which participates in vesicle formation that is associated with plant defense
(Hann & Rathjen, 2010). Type III effectors can also physically impede the function of their
target proteins (Feng & Zhou, 2012). For example, AvrPto and AvrPtoB interact with
BAK1 and inhibit the kinase activity of their targets (Shan et al., 2008). Post-translational
modification of host proteins is another common strategy employed by type III effectors
Introduction
7
(Feng & Zhou, 2012). For example, HopU1 ADP-ribosylates GRP7 on Arg47 and Arg49 to
abolish its RNA-binding activity and PTI function in plants. AvrAC and HopAI1 inhibit the
PTI signal transduction pathway by blocking phosphorylation of MPKs and BIK1.
Furthermore, multiple effector proteins have been shown to manipulate the JA pathway in
concert, such as AvrB, AvrRpt2, AvrPphB, HopPtoK, and AvrPphEpto (Chisholm et al.,
2006). Type III effectors can also target nucleic acids and regulate transcription of the host
plant. For example, XopD represses the expression of several defense-related genes in
tomato plants (Feng & Zhou, 2012) and AvrBs3 effector family of X. campestris alters
plant nuclear gene transcription, likely as a mean to down-regulate host defense (Chisholm
et al., 2006, Bonas & Van den Ackervaken, 1997).
The function of effectors is redundant and interchangeable. The redundancy of effectors is
illustrated by the combinatorial deletion of the 28 effectors of P. syringae pv. tomato
DC3000 which are collectively essential but individually dispensable for the ability of the
bacteria to defeat defenses, grow, and produce symptoms in plants (Kvitko et al., 2009).
Functional redundancy of effectors is achieved by targeting common host components
through different molecular strategies (Hann et al., 2010). Two effectors that illustrate
effector redundancy and interchangeability are the unrelated AvrPto and AvrPtoB proteins
of P. syringae pv tomato DC3000 (Pto DC3000) which both target the flagellin receptor
complex (Hann et al., 2010).
1.3.2 Translocation and function of oomycetes effectors
Oomycetes, are filamentous eukaryotes that are more closely related to brown algae than
fungi, cause some of the most devastating plant diseases such as late blight of potato and
sudden oak death. The identification of a common motif, RXLR, in oomycete AVR
proteins sparked excitement and speculation regarding translocation of effectors from these
fungus-like pathogens (Rehmany et al., 2005, Whisson et al., 2007). In plants, effectors can
be translocated into the host cell (cytoplasmic effectors) or targeted to the apoplast
(apoplastic effectors) (Bozkurt et al., 2012). Two large classes of cytoplasmic effectors,
RXLR and crinkler (CRN) proteins could be reliably predicted by the occurrence of the
conserved motifs in an N-terminal region that follows the signal peptide (Bozkurt et al.,
2012). In a noted yet controversial paper, Kale et al. proposed that binding of oomycete and
Introduction
8
fungal pathogen effectors to PI3P via the RXLR domain is required for host cell entry via
lipid raft-mediated endocytosis (Kale et al., 2010). However, this model has not been
universally accepted due to lack of reproducibility of the PI3P binding experiments and the
discovery that a conserved C-terminal domain, rather than the RXLR domain, of AVR3a is
required for binding to phosphatidylinositol monophosphates (PIPs) in vitro. Additionally,
recognition of PI3P in endoplasmic reticulum by the host-targeting signal of secreted
effectors of Plasmodium falciparum can facilitate export of the effector proteins from the
intracellular pathogen into the surrounding erythrocyte (Bhattacharjee et al., 2012).
Therefore, the mechanisms of RXLR effectors entry into plant cells remain unclear and
under debate (Bozkurt et al., 2012).
Many apoplastic effectors act as enzyme inhibitors, e.g. chitinases, glucanases and
proteases. For example, glucanase inhibitor proteins (GIPs) which are secreted by
Phytophthora sojae, specifically inhibit the endoglucanase activity of their plant host (Rose
et al., 2002). P. infestans deploys Kazal-like and cystatin protease inhibitors to target
secreted host serine and cysteine proteases, respectively (Tian et al., 2007, Tian et al.,
2004, Bozkurt et al., 2012). Recently, the P. infestans RXLR effector AVRblb2 has been
shown to localize to plasma membrane and prevent secretion of a plant immune protease at
the haustorial interface (Bozkurt et al., 2011). A second type of apoplastic effectors
interferes with adhesion, and possibly signalling, between host cell wall and plasma
membrane. For example, IPI-O of P. infestans can mediate disruption of plasma
membrane-cell wall contacts and interfere with cell-wall-associated defences (Stassen &
Van den Ackerveken, 2011). A third type of apoplastic effectors are toxins that are
produced by most necrotrophic or hemibiotrophic oomycetes. These include two families of
toxic proteins, PcF/SCR proteins and NEP1 like proteins (NLPs) (Stassen & Van den
Ackerveken, 2011).
1.3.3 Identification and function of fungal effectors
Most fungal avirulence genes encode virulence factors that are small secreted cysteine-rich
proteins with no homology to known proteins in the databases (Stergiopoulos & de Wit,
2009, Chisholm et al., 2006). Fungal effectors are also grouped into extracellular effectors
that are secreted into the apoplast or xylem of their host plants and cytoplasmic effectors
Introduction
9
that are translocated into host cells (Stergiopoulos & de Wit, 2009). Two rust cytoplasmic
effectors, RTP1 of Uromyces fabae and AvrP123 of M. lini, accumulate in the host nucleus
and may have a role in manipulating host gene expression during infection (Kemen et al.,
2005, Rafiqi et al., 2012). Two apoplastic effector proteins, Avr2 and Avr4, have been
characterized from the leaf-mold fungus Cladosporium fulvum (Chisholm et al., 2006).
Avr2 inhibits tomato Rcr3 cysteine protease required for Cf-2-dependent disease resistance
(Rooney et al., 2005). Avr4 contains a chitin binding domain that binds chitin and is
thought to shield the fungal cell wall from plant chitinases (van den Burg et al., 2003).
The sequencing of U. maydis genome identified 554 secreted proteins, among these 386
could not be ascribed a function including 272 that are either specific to U. maydis or
contain no recognisable domains (Kamper et al., 2006, Ellis et al., 2009). Of all the genes
encoding secreted proteins, 12 clusters of genes were found, which comprise 3-26 genes
and are scattered all over the genome. Deletion of individual clusters resulted in phenotypes
ranging from complete lack of symptoms to hypervirulence (Kamper et al., 2006).
Recently, a secreted chorismate mutase of U. maydis-Cmu1 was shown to be taken up by
plant cells and changes the metabolic status of host cells through metabolic priming
(Djamei et al., 2011). This is one of the few cases where effector function could be
suggested by amino acid sequence features (Rafiqi et al., 2012). Two extracellular effectors
of U. maydis, Pep1 and Pit2 are required for virulence of this fungus (Doehlemann et al.,
2011, Hemetsberger et al., 2012). Pep1 is shown to inhibit the peroxidase driven oxidative
burst and thereby suppress the early immune responses of maize. Although advances in
effectors identification have been made, the functional characterization of huge numbers of
novel effector proteins is significantly lags behind.
1.4 Co-evolution of plants and their microbial pathogens
Microbes have been interacting with plants for hundreds of millions of years. During co-
evolution of plants and their microbial pathogens, the plant and microorganism have
competing interests, which lead to an evolutionary ‘arms race’ in which the interaction
constantly selects for genetic changes in both pathogen and plant populations (Takken &
Rep, 2010). Genetic changes that enhance fitness, e.g. the ability to avoid host detection or
regain pathogen recognition ability, will be maintained in the population. The quantitative
Introduction
10
output of the plant immune system as well as the evolutionary relationship between PTI
and ETI was illustrated as a four phased ‘zigzag’ model (Fig. 2) (Jones & Dangl, 2006,
Dubery et al., 2012). In Phase 1, P/MAMPs are recognized by PRRs, resulting in PTI that
can stop further colonization. In Phase 2, successful pathogens deploy effectors that
contribute to pathogen virulence. Effectors can interfere with PTI. This results in effector-
triggered susceptibility (ETS). In phase 3, a given effector is ‘specifically recognized by
one of the NB-LRR proteins, resulting in effector-triggered immunity (ETI). In Phase 4,
natural selection drives pathogens to avoid ETI by shedding or diversifying the recognized
effector gene, or by acquiring effectors that suppress ETI. Thereafter, natural selection
results in the evolution of new R specificities so that ETI can be triggered again. Like all
models, the ‘Zig-Zag’ model could not explain every aspect of the host–pathogen
molecular interactions. This model has its limitations and fails to incorporate aspects of
damage-associated molecular patterns, necrotrophy and symbiosis, physical and temporal
scales, order of events and quantitative aspects of defenses (Pritchard & Birch, 2011).
1.5 Stp1 plays crucial role in the establishment of biotrophic interaction
between U. maydis and maize
Um02475 termed stp1 (stop after penetration 1) is one of the genes encoding secreted
effectors which is identified from the genomic sequencing and bioinformatic analysis of U.
maydis (Kamper et al., 2006, Schipper, 2009). stp1 is the rightmost gene in the three gene
cluster 5B, the deletion of which results in a complete loss of virulence symptoms in maize
infections (Kamper et al., 2006). Deletion analysis revealed that only stp1 is responsible for
the loss of virulence phenotype of a cluster 5B deletion mutant (Schipper, 2009). Homologs
of stp1 are found only in closely related smut fungi and bioinformatic analysis gave no
hints to functional domains.
Fungal growth of stp1 deletion mutant arrest directly after penetration of the first epidermal
cell and tumors were never found. Diaminobenzidine staining of infected leaves revealed
that H2O2 and phenolic compounds accumulate around penetration sites of stp1 deletion
mutants which indicated that a hypersensitive response was triggered. Microarray analysis
also illustrated the difference between SG200 and SG200∆stp1 in plant responses at
transcriptomic level. The strong plant defense response elicited by stp1 deletion mutants
Introduction
11
suggested that Stp1 was able to suppress plant defense reactions directly or indirectly.
Functional domain analyses of Stp1 revealed that the central glycine-rich domain of Stp1
was dispensible for protein function while the N- and C-terminal conserved domains were
essential during biotrophic growth. Confocal microscopy observation indicated that Stp1-
mCherry fusion proteins localized to the apoplastic interaction zone of infected plant cells.
After transient expression in Nicotiana benthamiana, Stp1 lacking the signal peptide
specifically localized to sub-compartments of the nucleus (Schipper, 2009).
Five intracellular plant proteins were identified to interact with Stp1 in a yeast-two hybrid
screen (Schipper, 2009). Moreover, one of the interactors, Sip12, co-localized with Stp1
lacking the signal peptide after expression in N. benthamiana (Schipper, 2009). These
Fig.2. A zigzag model illustrates the quantitative output of the plant immune system (Jones & Dangl, 2006). In this scheme, the ultimate amplitude of disease resistance or susceptibility is proportional to [PTI–ETS+ETI]. In phase 1, plants detect MAMPs/PAMPs (red diamonds) via PRRs to trigger PAMP-triggered immunity (PTI). In phase 2, successful pathogens deliver effectors that interfere with PTI, or otherwise enable pathogen nutrition and dispersal, resulting in effector-triggered susceptibility (ETS). In phase 3, one effector (indicated in red) is recognized by an NB-LRR protein, activating effector-triggered immunity (ETI), an amplified version of PTI that often passes a threshold for induction of hypersensitive cell death (HR). In phase 4, pathogen isolates are selected that have lost the red effector, and perhaps gained new effectors through horizontal gene flow (in blue)—these can help pathogens to suppress ETI. Selection favours new plant NB-LRR alleles that can recognize one of the newly acquired effectors, resulting again in ETI.
Introduction
12
results suggested that Stp1 might be translocated into the plant cell where it could be
involved in regulation of plant defense responses.
1.6 Aims of this study
The focus of this study is functional analysis of Stp1, a secreted effector that is crucial for
the establishment of biotrophic interaction between U. maydis and its host plant maize. The
main emphasis in this study is (1) identification of interactors of Stp1 through yeast two-
hybrid assay and analysis of the mechanism of the interaction to uncover the function of
Stp1; (2) determination whether Stp1 is an apoplastic or cytoplasmic effector; (3)
heterologous expression & purification of Stp1 and structural analysis of Stp1 to reveal
functional information residing in the structure; (4) functional domain analysis of Stp1.
Results
13
2. Results
2.1 Functional domain analysis of Stp1
2.1.1 The variable domains of Stp1 are dispensable
An amino acid sequence alignment of Stp1 orthologs shows that N- and C-termini of Stp1
are conserved among different smut fungi while the central glycine-rich domain is highly
divergent (Fig. 3). Previous experiments have already shown that Stp1 without part of the
glycine-rich region (Stp1Δ136-338) could complement SG200Δstp1 (Schipper, 2009). To
analyze whether the whole glycine-rich domain is dispensable or not, I generated a plasmid
p123pstp1-stp1Δ136-432 which contained a stp1 allele that retained only the coding region for
107 amino acids of the N-terminus and for 83 amino acids of the C-terminus (Fig. 4). This
plasmid was inserted into the ip locus (Loubradou et al., 2001) of SG200Δstp1 to produce
SG200∆stp1-stp1Δ136-432. Plant infection assays with three independently generated strains
showed that Stp1Δ136-432 could fully complement SG200Δstp1 (Fig. 5). This result shows
that Stp1Δ136-432 retains its function even though 58 % of the protein is deleted and
illustrates that the glycine-rich central domain of Stp1 is dispensable.
Fig. 3. Stp1 orthologs exist in related smut fungi. S. relianum: Sporisorium reilianum (Schirawski et al., 2010), U. scitamineum: Ustilago scitamineum (R. Kahmann, unpublished), U. hordei: Ustilago hordei (Laurie et al., 2012), U. maydis: Ustilago maydis (Kamper et al., 2006). Green color denotes conserved amino acids. Pink color denotes variable domains.
Results
14
Fig. 5. Stp1Δ136-432 complements SG200Δstp1. Strains tested for virulence in seedling infection are indicated. For each strain, three independent infections were performed and averaged. The total number of maize plants infected is given above each column. Symptoms were scored 12 days after infection following the scheme developed by Kamper et al (2006) indicated on the right.
Fig. 4. Domain structure of Stp1 and Stp1 mutant proteins. To test tumor formation, Stp1 derivatives were integrated into the ip locus of G200Δstp1 and the respective strains were used to infect maize plants. Stp1∆432-515 and Stp1∆40-136 were constructed by Kerstin Schipper (Schipper, 2009). Red (SP): signal peptide, grey: variable domains of Stp1, orange: N-terminus of Stp1, blue: C-terminus of Stp1.
Results
15
To rule out the possibility that the central domain of Stp1 might have a minor function, a
plasmid p123pstp1-Stp1∆29-136/432-515 in which the variable glycine-rich domain was fused
with the signal peptide was constructed (Fig. 4) and inserted into the ip locus of
SG200∆stp1 to generate SG200∆stp1-stp1∆29-136/432-515. No disease symptoms were detected
on plants infected with SG200∆stp1-stp1∆29-136/432-515 (Fig. 6). To detect partial
complementation, SG200∆stp1-stp1∆29-136/432-515 infected maize leaves were stained with
WGA-AF 488 / propidium iodide (WGA/PI) and observed by confocal microscopy. Similar
to the stp1 deletion mutant, growth of SG200∆stp1-stp1∆29-136/432-515 stopped after
penetration. Occasionally, some hyphae of SG200∆stp1-stp1∆29-136/432-515 were proliferating
in vascular bundles (Fig. 7 A and D). The hyphae of SG200∆stp1 were also observed in
vascular bundles in rare cases (Fig. 7 B and E). But, SG200∆stp1-stp1∆29-136/432-515 which
proliferate along the vascular bundles in a continuous way grew better than SG200∆stp1
and triggered less plant defense responses than SG200∆stp1 (Fig. 7). This could indicate
that the expression of the glycine-rich domain of stp1 could have a weak effect on
Fig. 6. N- and C-termini of Stp1 can be expressed separately while the strains expressing glycine-rich domain of Stp1 cannot restore tumor formation. Strains tested for virulence in seedling infection are indicated. The total number of maize plants infected is given above each column. Symptoms were scored 12 days after infection following the scheme developed by Kamper et al (2006) indicated on the right.
Results
16
biotrophic development. However, in very rare case, the hyphae of SG200 could also be
observed in vascular bundle (Fig. 7 C and F). Therefore, further studies are needed to
substantiate this point quantitatively.
Furthermore, the complementation of SG200∆stp1 by N-terminus truncated Stp1 indicated
that the segment spanning amino acids 29-55 of Stp1 is dispensable (Fig. 4) (Schipper,
2009). To further analyze the functional regions close to the C-terminus, the plasmids
p123pstp1-stp1∆433-454, p123pstp1-stp1∆455-476, p123pstp1-stp1∆477-494 and p123pstp1-
stp1∆495-515 incorporating C-terminus truncated stp1 genes were constructed and kindly
provided by K. schipper. All plasmids were inserted into SG200Δstp1 to produce
SG200∆stp1-stp1Δ433-454, SG200∆stp1-stp1Δ455-476, SG200∆stp1-stp1Δ477-494 and
SG200∆stp1-stp1Δ495-515. Of these strains, only SG200∆stp1-stp1Δ455-476 was able to cause
Fig. 7. SG200∆stp1-stp1∆29-136/432-515 proliferation in vascular bundles. A, B and C: Maize plants infected with indicated strains. D, E and F: Enlargement of the areae in the white rectangular of A, B and C. Maize leaves infected with indicated strains were collected one day after infection, stained with WGA/PI as described in methods and observed using confocal microscopy. The pictures are the overlay of WGA and PI channels. The Fungal hyphae are shown in green. The plant cell wall, vascular bundle and the autofluorescence indicating plant defense responses are shown in red. Yellow colored areae are the overlap of the signals of fungal hyphae and plant defense responses. The white arrows denote the red ring of vascular bundles. The yellow arrows denote the plant defense responses.
Results
17
disease in preliminary test, which demonstrated that the Stp1 segment between amino acids
455 and 476 was dispensable (Fig. 8). The infection will be repeated to substantiate this
result.
2.1.2 N- and C-termini of Stp1 could be separately expressed
To elucidate whether the two domains of Stp1 can be expressed as separate proteins, a
plasmid p123pstp1-stp1∆137-515+pstp1-stp1∆37-431 that specifies two gene fragments encoding
N- and C-terminus of Stp1, both fused with the stp1 promoter and signal peptide was
constructed and kindly provided by M. Daume and K. Schipper (Fig. 4). This plasmid was
inserted into the ip locus of SG200∆stp1 to produce SG200∆stp1-stp1∆137-515+stp1∆37-431.
The plant infection assays of this strain demonstrated that separately expressed N- and C-
termini of Stp1 were functional (Fig. 6).
No tumors were formed on plants infected with SG200∆stp1-stp1∆432-515 or SG200∆stp1-
stp1∆40-136 (Fig. 4) in which either N- or C-termini of Stp1 are deleted (Schipper, 2009).
This had indicated that both N- and C-terminus of Stp1 are needed for its function
Fig. 8. Stp1 segment between amino acids 455 and 476 is dispensable. Strains tested for virulence in seedling infection are indicated. The total number of maize plants infected is given above each column. Symptoms were scored 12 days after infection following the scheme developed by Kamper et al (2006) indicated on the right.
Results
18
(Schipper, 2009). To investigate whether expression of only the N- or the C-terminal
domain of Stp1 is sufficient to allow partial colonization, maize leaves infected with
respective strains were stained with WGA/PI and observed using confocal microscopy. The
results showed that SG200∆stp1 stopped after penetration while SG200 profusely
colonized the tissue (Fig. 9 panel A). Meanwhile, colonization of SG200∆stp1-stp1∆432-515
and SG200∆stp1-stp1∆40-136 also stopped after penetration (Fig. 9 panel A). Besides the
representative growth of indicated U. maydis strains in panel A (Fig. 9), some hyphae of
SG200∆stp1 could proliferate on plant surface which could not reflect colonization (Fig. 9
panel B). Additionally, SG200∆stp1, SG200∆stp1-stp1∆432-515 and SG200∆stp1-stp1∆40-136
were detected in deeper tissue layers (Fig. 9 panel B). However, it is difficult to determine
whether the deeper growth reached mesophyll cells or it ceased in epidermal cells. So far,
no obvious differences between SG200∆stp1, SG200∆stp1-stp1∆432-515 and SG200∆stp1-
stp1∆40-136 were observed.
To further visualize leaf colonization, Chlorazol black E was used for staining. SG200
proliferates in both epidermal tissue and mesophyll tissue (Fig. 10 A, E). Most hyphae of
Fig. 9. Early biotrophic development of SG200∆stp1:stp1∆432-515 and SG200∆stp1:stp1∆40-136. The maize leaves infected with indicated U. maydis strains were collected three days after infection, stained with WGA/PI as described in methods and observed using confocal microscopy. The first figure in panel B showed surface growth of SG200∆stp1. The pictures shown are the overlay of WGA and PI channels. The fungal hyphae are shown in green. The plant cell walls as well as the autofluorescence indicating plant defense responses are shown in red. The white arrows denote appresoria.
Results
19
SG200∆stp1 as well as SG200∆stp1-stp1∆432-515 and SG200∆stp1-stp1∆40-136 stopped
growing immediately after penetration as shown in Fig. 9. Rarely, hyphae of SG200∆stp1-
stp1∆432-515 and SG200∆stp1-stp1∆40-136 could penetrate epidermal cells and grow into
mesophyll cells (Fig. 10 G, H). However growth inside mesophyll cells could also be rarely
observed in SG200∆stp1 infected plants (Fig. 10 F). The finding that neither the N- nor the
C-terminus of Stp1 can partially rescue the growth arrest of the stp1 mutant indicated that
both N- and C-terminus of Stp1 were playing crucial functional roles. Further studies need
to be performed to determine if there are differences between SG200∆stp1, SG200∆stp1-
stp1∆432-515 and SG200∆stp1-stp1∆40-136 in terms of the ratio of fungal hyphae penetrated
into mesophyll cells or the length of fungal hyphae.
2.1.3 Stp1 related proteins in other smut fungi can replace Stp1 of U. maydis
N- and C-termini of Stp1 are conserved among different smut fungi while the middle
central glycine-rich domain is highly divergent (Fig. 3). To determine whether these
orthologs can substitute for Stp1 in U. maydis, we generated the plasmids p123pstp1- Uh-
stp1 and p123pstp1-Us-stp1 in which stp1 orthologs from U. hordei and U. scitamineum
Fig. 10. Plant colonization by SG200∆stp1-stp1∆432-515 and SG200∆stp1-stp1∆40-136. The maize leaves infected with indicated U. maydis strains were collected two days after infection, stained with chlorazol black E as described in methods and observed using confocal microscopy at bright field. Two figures in the same red rectangular denote two different cross sections of the same sample. The black arrows in B, C and D denote appresoria, in F G and H denote the fungal hyphae in mesophyll and in A and E denote branching of fungal hyphae in both epidermal and mesophyll.
Results
20
were fused with the promoter and signal peptide of stp1 from U. maydis respectively. The
plasmids were inserted into SG200∆stp1 to produce SG200∆stp1-Uh-stp1 and
SG200∆stp1-Us-stp1. The plant infection assays (Fig. 11) demonstrated that both stp1
orthologs from U. hordei and U. scitaminem could complement SG200∆stp1 (Fig. 11)
which indicated that the function of Stp1 orthologs was conserved.
2.1.4 The putative functional domains show low similarity with known functional
domains in the databases are not valid
BLAST analysis indicated that the C-terminus of Stp1 showed low similarity with the
NAD+-binding domain of NmrA (nitrogen metabolism repression regulator NmrA) family
proteins (Nunez-Corcuera et al., 2008). Therefore, the plasmids P123pstp1-stp1Δ136-432T452V
and P123pstp1-stp1T452V in which the threonine 452 was replaced by valine were
constructed to test if they could complement SG200Δstp1. Both plasmids were introduced
into SG200Δstp1 respectively to produce SG200∆stp1-stp1Δ136-432T452V and SG200∆stp1-
stp1T452V. Plant infection assays showed that both SG200∆stp1-stp1Δ136-432T452V and
Fig. 11. stp1 orthologs from other smut fungi can replace stp1 of U. maydis. Strains tested for virulence in seedling infection are indicated. The total number of maize plants infected is given above each column. Symptoms were scored 12 days after infection following the scheme developed by Kamper et al (2006) indicated on the right.
Results
21
SG200∆stp1-stp1T452V could cause disease on maize plants (Fig. 12), which indicated that
the presumed NAD+-binding function of Stp1 might not be relevant for its function.
The very C-terminal domain of the Tin2 effector plays an important functional role (S.
Tanaka, personal communication). To test whether the very C-terminus of Stp1 was
essential or not, the plasmids p123pstp1-stp1PPAA and p123pstp1-stp1SRAA, in which the C-
terminal PP and SR were replaced by AA respectively, were generated. Both plasmids were
introduced into SG200Δstp1 to produce SG200∆stp1-stp1PPAA and SG200∆stp1-stp1SRAA.
Plant infection assays revealed that both SG200∆stp1-stp1PPAA and SG200∆stp1-stp1SRAA
could cause disease on maize plants (Fig. 13), which indicated that the very C-terminus of
Stp1 was not crucial for its function.
Fig. 12. The putative NAD+-binding domain of Stp1 is not relevant for its function. Strains tested for virulence in seedling infection are indicated. The total number of maize plants infected is given above each column. Symptoms were scored 12 days after infection following the scheme developed by Kamper et al (2006) indicated on the right.
Results
22
2.2 Interactors of Stp1 and Stp1∆136-432 are both cytoplasmic and apoplastic
maize proteins
To answer what host protein (s) is being targeted leading to enhanced virulence, one of the
most common way is to identify host proteins that physically interact with an effector.
Among several methods exist, the most frequently utilized is yeast two-hybrid system
(Munkvold & Martin, 2009). To screen for interactors of Stp1, yeast two-hybrid assays had
been firstly performed using full-length Stp1 as bait (Schipper, 2009) and then redone using
Stp1∆136-432 as bait in this study.
2.2.1 Interactors identified by full-length Stp1 are not likely to be functionally
relevant
Employing full-length Stp1 as bait, five putative interaction partners of Stp1 (Sip2-
adenylate kinase Adk1, Sip10-homolog to myrosinase precursor, Sip12-RING-E3 ubiquitin
ligase, homolog to Vip2, Sip29-potential transcription factor and Sip31-GroEL chaperone)
Fig. 13. The very C-terminus of Stp1 is not critical for its funcion. Strains tested for virulence in seedling infection are indicated. The total number of maize plants infected is given above each column. Symptoms were scored 12 days after infection following the scheme developed by Kamper et al (2006) indicated on the right.
Results
23
had been identified through Y2H assays (Schipper, 2009). Meanwhile, deletion analysis
showed that the long glycine-rich domain in the middle of Stp1 was dispensable. To
determine whether the putative interactors were interacting specifically with the functional
N- or C- terminal domains, I have first tested the interaction between Stp1Δ136-338 and the
putative interaction partners-Sip12 and Sip29. The respective prey plasmids were kindly
provided by K. Schipper and the interactions were tested by yeast re-transformation. The
results showed that the truncated Stp1Δ136-338 was interacting with full-length Stp1 and
weakly with Sip12 (Fig. 14). However, Stp1Δ136-338 was not interacting with Sip29 (Fig.
14). To test the interactions between Stp1Δ136-432 and Sip10, Sip12, Sip29 and Sip31,
pGBK-stp1∆136-432 was generated and the corresponding plasmids kindly provided by K.
Schipper were co-transformed into AH109. The results showed that Stp1Δ136-432 was
interacting with full-length Stp1, but it was not interacting with any of the putative
interaction partners of the full-length Stp1 (Fig. 14). In the expression assays using Western
blotting, the expression of AD-Sip12 (AD, activation domains of GAL4) and AD-Sip29
were not detected (Fig. 14). Full-length sip12 and sip29 will be cloned and the interactions
will be retested. The Y2H assays indicated that the interaction between Stp1 and Sip10,
Sip12, Sip29 and Sip31 was not likely to be functionally relevant. Therefore, Y2H assays
were redone employing Stp1∆136-432 as bait.
2.2.2 Interactors of Stp1∆136-432 are both cytoplasmic and apoplastic maize proteins
pGBK-stp1∆136-432 and a prey cDNA library of U. maydis infected maize leaves (two days
and five days post infection) were subsequently co-transformed into AH109. Duplicates
were eliminated by sorting after PCR amplification and digestion by HaeIII.
After screening of the library, twelve distinct interaction partners of Stp1∆136-432 were
identified and verified by re-transformation (Table. 1). Bioinformatic analysis by Signal P
(http://www.cbs.dtu.dk/services/SignalP/) and TargetP
(http://www.cbs.dtu.dk/services/TargetP/) showed Sip1 and Sip3 were predicted to be
secreted maize proteins while the others were predicted to be cytoplasmic proteins (Table
1). Interestingly, although the interactors identified by full-length Stp1 are not interacting
with Stp1∆136-432, all putative interactors identified using Stp1∆136-432 as bait were interacting
Results
24
Fig. 14. Sip10, Sip12, Sip29 and Sip31 are not interacting with Stp1Δ136-432 in Y2H assays. A, Interactions between Sip12, Sip29 and StpΔ136-338. B, Interactions between Sip10, Sip12, Sip29, Sip31 and StpΔ136-432. The re-transformation and growth assay were performed as described in methods. C, Expression of indicated proteins in yeast was detected by Western blotting. DNA binding domains (BD) were detected using c-Myc antibody and activation domains (AD) were detected using HA antibody. The red arrows denote the signals of indicated proteins (BD-Stp1: 68.5 kDa, BD-StpΔ136-432: 42 kDa, AD-Sip10: 72.7 kDa, AD-Sip12: 54.8 kDa, AD-Sip29: 123.1 kDa, AD-Sip31: 42 kDa, AD-Stp1: 68.5 kDa).
Results
25
Interaction partners Signal peptide
FrequencyInteraction with Stp1∆136-432
Interaction with Stp1
Sip1 Putative beta-galactosidase BG1 Yes 1 N N
Sip2 Putative Adenylate kinase No 8 N +
Sip3▲ Putative cysteine protease 1 Yes 1 + +
Sip4 Putative chaperone protein dnaJ No 156 + N
Sip5 Putative DAG protein No 1 + N
Sip6▲ Putative Rhamnogalacturonate lyase No 379 - +
Sip7 Hypothetical protein No 54* + +
Sip8▲ Putative thiamine biosynthesis protein thiC No - +
Sip9▲ Putative SAT5 (cell number regulator 8) No 138 + +
Sip10 Putative myrosinase precursor No 15 - +
Sip11 Putative Transducin family protein No 2 + N
Sip12 Putative VIP2 (E3 ubiquitin ligase) No 45 N +
Sip14▲ Putative VIP2 (E3 ubiquitin ligase) No 1 - +
Sip16▲ Putative CCR4-NOT transcription complex subunit No 1 + +
Sip17 Putative iron-sulfur protein2 No 2 N N
Sip19▲ Putative Serine/threonine-protein kinase MHK No 2 + +
Sip20 Putative FPA RNA binding No 3 N N
Sip21▲ Putative VIP2 No 1 + +
Sip29 Putative transcription factor No 37 - +
Sip31 Putative GroEL chaperone No 3 - +
with full-length Stp1 (Fig. 14 and Table. 1) which indicated that the glycine-rich domain of
Stp1 was binding unspecifically with many different proteins.
In many cases, the putative interactors isolated from the library contained only a fragment
of the full-length gene. Therefore, before further analysis, I chose eight putative interactors
(Sip3, Sip6, Sip8, Sip9, Sip14, Sip16, Sip19 and sip21) for the interaction studies depicted
in Table 1, which could conceivably be involved in plant defense reaction or plant
development regulation. Full-length genes of them were cloned from cDNA of U. maydis
infected maize leaves. These genes were then inserted into pGADT7 vector to generate
pGAD-sip3, pGAD-sip6, pGAD-sip8, pGAD-sip9, pGAD-sip14, pGAD-sip16, pGAD-
Table. 1. Y2H interaction partners of Stp1 and Stp1Δ136-432 are both apoplastic and cytoplasmic maize
proteins.
N: not tested yet, *: Both the size of PCR products and the pattern of HaeIII digestion fragments of Sip7 and Sip8 are identical. Therefore, their frequency could not be separated. ▲: interaction was tested with full-length cDNA clones of the respective genes. Previously identified interaction partners (Schipper, 2009) are indicated in red fonts
Results
26
sip19 and pGAD-sip21. Their interactions with Stp1 and Stp1∆136-432 were then tested by
co-transformation. The results showed that Sip6, Sip8 and Sip14 were not interacting with
Stp1∆136-432 anymore (Fig. 15). 50.2 % isolates from Y2H screening is Sip6, the putative
rhamnogalacturonate lyase, and most of the isolates is C-terminus of rhamnogalacturonate
lyase. This indicates that one important source of false positives is the gene fragments.
Sip14 and Sip21 are both putative VIP2 proteins which is a component of transcription
complex units. The difference between these two proteins is that in Sip21 the N-terminal
Ringfinger domain is missing which indicated that the interaction between Sip21 and Stp1
was not likely correlated with the Ringfinger domain.
2.2.3 N- and C-termini of Stp1 may play separate functions
To learn via which domain Stp1 was binding to the interactors, pGBK-stp129-135 and
pGBK-stp1433-515 were generated. The interactions between the full-length maize proteins
and separate N- or C-terminus of Stp1 were also tested by co-transformation. The results
showed that Sip6 was only weakly interacting with N-terminus of Stp1 while the other
interactors were interacting strongly with N-terminus (Fig. 15). Sip21 was interacting
weakly with the C-terminus of Stp1, Sip8 and Sip14 were not interacting with the C-
terminus of Stp1 and the other interactors were interacting strongly with the C-terminus of
Stp1 (Fig. 15). Whether the interaction between Sip6 and C-terminus of Stp1 or Sip8 &
Sip14 and N-terminus of Stp1 reflect transient or weak interaction between Stp1∆136-432 and
Sip6, Sip8 and Sip14 or they are not functional relevant still need to be tested
biochemically. The differences observed suggested that N- and C-termini of Stp1 may play
distinct functions. To investigate the relationship between different domains of Stp1,
pGAD-stp1∆136-432, pGAD-stp129-135 and pGAD-stp1433-515 were constructed. The
interactions between different domains of Stp1 were tested using Y2H assays. The results
showed that Stp1 was interacting with itself as well as Stp1∆136-432 while Stp1∆136-432 was
not interacting with itself (Fig. 16). The C-terminus of Stp1 (Stp1433-515) was interacting
with itself weakly. The N-terminus of Stp1 (Stp129-135) showed no self-interaction and was
unable to interact with the C-terminus (Fig. 16). This could indicate that N- and C-termini
of Stp1 were not functioning in a complex.
Results
27
Fig. 15. Stp1 mutants were interacting with interactors isolated using Stp1Δ136-432 as bait with different affinity. A, Full- length Sip6, Sip8 and Sip14 do not interact with Stp1Δ136-432 in Y2H assays. B, N- and C-termini of Stp1 showed different patterns of interaction from that of Stp1Δ136-432 and Stp1. The re-transformation and growth assay were performed as described in methods. C, Expression of indicated proteins in yeast detected by Western blotting. BD was detected using c-Myc antibody and AD was detected using HA antibody. The red arrows denote the signals of indicated proteins (BD-Stp129-135: 33.9 kDa, BD-Stp433-515: 29.5 kDa. AD-Sip16: 54 kDa, AD-Sip9: 48.6 kDa, AD-Sip8: 95.1 kDa, AD-Sip19: 72.2 kDa, AD-Sip21: 60.3 kDa).
Fig. 16. Interaction between different domains of Stp1 tested by Y2H assays. The re-transformation and growth assays were performed as described in methods.
BA C
Results
28
2.2.4 Stp1 can interact with several cysteine proteases of maize
One of the interaction partners of Stp1 is Sip3 encoding maize cysteine protease predicted
to be secreted (Table. 1). Recently, Five distinct cysteine proteases (CP1-like A, CP1-like B
(Sip3), CP2-like, XCP2 and CatB3-like) were identified from apoplastic fluid of U.maydis
infected maize leaves (van der Linde et al., 2012). The identification of Sip3 in apoplastic
fluid verified that Sip3 was an extracellular cysteine protease of maize. In addition, another
apoplastic maize cysteine protease, Mir3, was identified as interactor of the Tin2 effector
(N. Neidig, personal communication). To determine whether Stp1∆136-432 was binding
specifically to Sip3 or binding to all cysteine proteases identified, the interactions between
Stp1∆136-432 and Sip3 (CP1-like B), Mir3 (CP1-like A), CP2-like, XCP2-like and CatB3-like
were tested (respective prey plasmids were kindly provided by N. Neidig and A. Müller).
The results showed that Stp1∆136-432 was interacting with all tested cysteine proteases with
different affinity (Fig. 17). This indicated that the Stp1∆136-432 may be interacting with this
class of cysteine proteases.
Fig. 17. Stp1∆136-432 interacts with a class of cysteine proteases. A, Interaction between Stp1∆136-432 and cysteine proteases. The re-transformation and growth assay were performed as described in methods. B, Expression of indicated proteins in yeast detected by Western blotting. BD was detected using c-Myc antibody and AD was detected using HA antibody. The red arrows denote the signals of indicated proteins (AD-Sip3: 72.7 kDa, AD-Mir3: 67.5 kDa, AD-CP1A: 56.7 kDa, AD-CP2: 44.1 kDa, AD-XCP2: 44 kDa, AD-CatB3: 47.8 kDa).
A B
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29
2.2.5 Comparison of the interactors with microarray data
10 interactors of Stp1 could be identified in the transcriptomes assessed by microarray
(Table. 2) (Skibbe et al., 2010). When we adopt the same threshold with RNA-Seq analysis
(Fold change=2, P-value=0.01, Table. 2), Sip4 and Sip17 were slightly up-regulated in
seedling (1 dpi) while Sip8 and Sip17 were slightly down-regulated in adult leaves (Sip8-9
dpi, Sip 17-3 dpi) and Sip17 was obviously down-regulated in adult leaves (9 dpi).
However, all the other interactors especially Sip3, Sip9, Sip16, Sip19 and Sip21 which
were confirmed to interact with Stp1∆136-432 by full-length cDNA clones were not
differentially expressed. In addition, full-length Sip8 was not interacting with Stp1∆136-432
while the interactions between Sip4, Sip17 and Stp1∆136-432 still need to be confirmed by
cloning full-length genes. Therefore, most interactors isolated by Y2H are not differentially
expressed significantly in microarray analysis.
Adult leaf 3 dpi Adult leaf 9 dpi Seedling 1 dpi Seedling 3 dpi
P-value Foldch P-value Foldch P-value Foldch P-value Foldch
Sip3 0.168 0.80 0.004 0.58 0.926 1.02 0.046 0.71
Sip4 0.0607 0.64 0.405 1.24 0.0068 2.19 0.171 0.73
Sip8 0.00061 0.59 0.000089 0.42 0.105 1.23 0.0001 0.54
Sip9 0.484 0.95 0 1.62 0.814 0.98 0.092 0.87
Sip12 0.911 1.03 0.658 1.1 0.675 1.54 0.58 0.88
Sip14 0.312 0.81 0.67 1.09 0.025 1.66 0.245 0.78
Sip16 0.597 0.91 0.0046 0.56 0.027 1.52 0.541 0.90
Sip17 0.004 0.44 0 0.19 0.0012 2.01 0.049 0.60
Sip18 0.062 1.26 0.031 1.31 0.751 0.96 0.0002 1.74
Sip19 0.07 0.76 0.275 0.80 0.135 1.26 0.0052 0.62
2.3 Purification of recombinant Stp1 protein
2.3.1 Purification of His-Stp1∆136-432
To analyze the structure of Stp1 and characterize the interaction between Stp1 and its
interactors, Stp1∆136-432 was heterologously expressed in E. coli and purified. To this end,
pRSET His TEV Stp1∆136-432 was constructed and transformed into E. coli BL21. Large
Table. 2. Most of the inteactors isolated by Y2H are not identified among the differentially expressed
genes in microarray analysis (Skibbe et al., 2010)
Foldch: Fold change. The red fonts denote differential expression.
Results
30
amounts of His-Stp1∆136-432 (24.4 kDa) could be expressed in BL21, but most of the protein
was insoluble and ended up in the pellet faction (Fig. 18, Lane 2).
To increase the amount of soluble protein, I altered the temperature, the IPTG
concentration and added detergents like Triton X-100 (not shown). However, there was no
significant improvement of amounts of Stp1 in the soluble fraction. To improve the
solubility, SoluBL21™ strain (BioCat), Champion™ pET SUMO Expression System,
GST-tag and codon optimized Stp1∆136-432 for E. coli (GENEART) were also tested, but
again without obvious improvement. Therefore, His-Stp1∆136-432 expressed in E. coli BL21
was used for protein purification using Ni-NTA-agarose. After Ni-NTA-agarose
purification, the abundance of His-Stp1∆136-432 was largely increased, but the purity of the
protein was still low (Fig. 18, Lane 3). To improve the purification, a more specific tag,
Strep-tag was fused to Stp1 and the protein was purified using Strep-Tactin Resins (IBA).
Additionally, dissociation buffers containing ATP, K+, and Mg2+ were also tested.
However, the purity of the protein could not be improved significantly (data not shown). To
purify Stp1∆136-432, I have chosen His-Stp1∆136-432 and scaled up the culture volume to at
least 6 litters. The protein eluted from Ni-NTA-agarose was then applied to a MonoQ
column. The purity after elution from the MonoQ column was greatly improved (Fig. 18,
Lane 4). Although there were still some contaminating proteins, the protein purified from
MonoQ was considered good enough for biochemical assays. However, to analyze the
6 7
Fig. 18. Purificaiton of His- Stp1∆136-432. Lane 1, Supernatant of pRSET His TEV Stp1∆136-432; Lane 2, Pellet of pRSET His TEV Stp1∆136-432; Lane 3, His-Stp1∆136-432 purified through Ni-NTA-agarose; Lane 4, His-Stp1∆136-432 purified through MonoQ column; Lane 5, His-Stp1∆136-432 purified through Superdex 75 column; Lane 6, Western blotting after gel filtration, His-Stp1∆136-432 was detected using Stp1 antibody (Schipper, 2009); Lane 7, SDS-PAGE corresponding to the Western blotting. Pageruler: PageRuler™ Prestained Protein Ladder SM0671. The red arrows showed Stp1∆136-432. The differences in the migration rate were caused by the different concentration of SDS-PAGE gels.
Results
31
structure of Stp1 by crystallography, the purity of His-Stp1∆136-432 from MonoQ needed to
be improved. Thus, it was applied onto Superdex 75 column (GE Healthcare). The purity of
Stp1∆136-432 after gel filtration was higher than 90 % analyzed by ImageQuant TL™
(TotalLab). After gel filtration, the bind of His-Stp1∆136-432 on SDA-PAGE (Fig. 18, Lane 5)
was confirmed by Western blotting (Fig. 18, Lane 6 and 7). To get rid of His tag, His-
Stp1∆136-432 was cleavage by TEV protease. The cleavage was not efficient (Fig. 19).
Therefore, Stp1∆136-432 protein carry an N-terminal His-tag was used for subsequent
experiments.
To analyze the purity and the confirmation features of His-Stp1∆136-432, besides SDS-PAGE
visualization, gel filtration and limited proteolysis was used. Gel filtration showed that His-
Stp1∆136-432 migrated in one main peak which also generated one single bind in SDS-PAGE
(Fig. 20). The small peak running in front of the main peak, which is probably
contaminating proteins or dimmers of His-Stp1∆136-432, could not be detected by SDS-PAGE
(Fig. 20). This indicates that His-Stp1∆136-432 visualized on SDS-PAGE after gel filtration is
pure protein (Fig. 18). The conformation features of His-Stp1∆136-432 were analyzed by
limited proteolysis assays (Fontana et al., 2004). The digestion by chymotrypsin showed
that His-Stp1∆136-432 without denaturation by SDS was more resistance to protease than
denatured protein (Fig. 21). In addition, the digestion of His-Stp1∆136-432 without
denaturation is increasing with the increase of the concentration of chymotrypsin (Fig. 21).
This indicates His-Stp1∆136-432 may have a correctly folded structure. In addition, His-
Stp1∆136-432 could be concentrated to at least 20 mg per ml in low salt buffer. It could also
be stored at room temperature for at least two weeks without obvious degradation (data not
shown). Finally, 10 mg (measured by protein quantificaion assays according to Bradford
Fig. 19. The cleavage of His- Stp1∆136-432 by TEV protease decreased the homogeneity of the protein. Lane 1, His-Stp1∆136-432 purified through Superdex 75; Lane 2, Stp1∆136-432 cleavaged from Ni-NTA agarose; Lane 3, His-Stp1∆136-432 eluted from Ni-NTA agarose. Pageruler: PageRuler™ Prestained Protein Ladder SM0671. The red arrows showed His-Stp1∆136-432. The red cycle denotes TEV-protease with a HQ tag.
Results
32
(Bradford, 1976)) His-Stp1Δ136-432 was purified. Crystallography is in progress in
collaboration with Michael Groll (TUM).
2.3.2 Purification of His-Stp129-135, His-Stp1433-515 and His-Aro7
Deletion analysis and Y2H assays suggested that N- and C-termini of Stp1 may play
distinct functions during establishment of the biotrophic interaction between U. maydis and
maize. To functionally analyze N- and C-termini of Stp1, both of them were also
heterologously expressed in E. coli. To express N- and C-termini of Stp1, pRSET His TEV
Stp129-135 and pRSET His TEV Stp1433-515 were generated and transformed into E. coli
Fig. 20. Gel filtration assay of His-Stp1Δ136-432. Left panel, gel filtration of His--Stp1Δ136-432. Right panel, SDS-PAGE of the peaks in gel filtration. The lane numbers, 15-23 are corresponding to the fractions in the gel filtration chart. The red arrow shows the peak of His-Stp1Δ136-432. X-axis, fractions and elution volume. Y-axis, UV abosrbance (mAU). Pageruler: PageRuler™ Prestained Protein Ladder SM0671.
Fig. 21. Limited proteolysis assay of His-Stp1∆136-432. Lane 1, Pageruler; lane 2, His-Stp1Δ136-432, 1.6 µg/ml chymotrypsin; lane 3, His-Stp1Δ136-432, 8 µg/ml chymotrypsin; lane 4, His-Stp1Δ136-
432, 40 µg/ml chymotrypsin; lane 5, His-Stp1Δ136-432, 1.6 µg/ml chymotrypsin plus 0.1 % SDS; lane 6, His-Stp1Δ136-432, 8 µg/ml chymotrypsin plus 0.1 % SDS; lane 7, His-Stp1Δ136-432, 40 µg/ml chymotrypsin plus 0.1 % SDS; His-Stp1Δ136-432 was digested by different concentration of chymotrypsin for 30 min at 30 oC. Pageruler: PageRuler™ Prestained Protein Ladder SM0671.
Results
33
BL21. After induction, both His-Stp129-135 (14.8 kDa) and His-Stp1433-515 (12.5 kDa) were
well expressed as His-Stp∆136-432 but most of the protein was insoluble (data not shown).
After optimization of expression conditions (See materials and methods), both His-Stp129-
135 and His-Stp1433-515 were purified employing Ni-NTA-agarose. This was followed by
chromatography on a MonoQ column. The purity of His-Stp129-135 was still very low which
precluded the following functional analysis of N-terminal domain of Stp1 (Fig. 22 B).
Additionally, the purity of His-Stp129-135 after MonoQ purification was even lower than the
purity after Ni-NTA purification which suggested that His-Stp129-135 was unstable (Fig. 22
A and B). His-Stp1433-515 did not bind to the MonoQ column while most contaminating
proteins remained bound to the column so that His-Stp1433-515 could be efficiently separated
from contaminant proteins. The flow through fraction of His-Stp1433-515 from MonoQ
column was collected and loaded on SDS-PAGE (Fig. 22 B). Besides Stp1 mutants, Aro7
the cytosolic chorismate mutase from U. maydis was also heterologously expressed and
purified as a negative control. To express Aro7, pRSET His TEV Aro7 was generated and
transformed into E. coli BL21. After Ni-NTA-agarose and MonoQ column purification,
His-Aro7 was prepared for the following biochemical assays (Fig. 22 B).
Fig. 22. Purificaiton of His-Stp129-135 , His-Stp1433-515 and His-Aro7. A, SDS-PAGE of His-Stp129-135 and His-Stp1433-515 after Ni-NTA agarose purification. B, SDS-PAGE of His-Stp129-135, His-Stp1433-515 and Aro7 after MonoQ purification. C, Western blotting of His-Stp129-135 and His-Stp1433-515 after MonoQ purification. Lane 1, His-Stp129-135 purified by Ni-NTA agarose; Lane 2, His-Stp1433-515 purified by Ni-NTA agarose; Lane 3, His-Aro7 purified by MonoQ; Lane 4, Main peak of His-Stp129-135 eluted from MonoQ column; Lane 5, Main peak of His-Stp1433-515 eluted from MonoQ column; Lane 6, Flowthrough of Stp129-135 from MonoQ column; Lane 7, Flowthrough of His-Stp1433-515 from MonoQ column; Lane 8-11 are corresponding western blotting to Lane 4-7. Pageruler, unstained Low Range Protein Ladder for A and B and PageRuler™ Prestained Protein Ladder SM0671 for C. The red cycles denote His-Stp129-135 and His-Stp1433-515 on SDS-PAGE. The differences in the migration rate were caused by the different concentration of SDS-PAGE gels.
Results
34
2.3.3 Purification of Strep-Sip3.
To study the interaction between Stp1 and Sip3 biochemically, Sip3∆351-469 (Sip3 without
granulin domain) was heterologously expressed through agrobacterium mediated transient
expression in tobacco. To express Sip3∆351-469 in tobacco, pBIN Strep sip3∆351-469 was
generated, transformed into A. tumefaciens and the transformant was infiltrated into young
leaves of N. benthamiana. Strep-Sip3∆351-469 was then purified employing IgG beads. The
amount of Strep-Sip3∆351-469 purified from N. benthamiana was too low to be detected by
SDS-PAGE (not shown). The enzymatic activity of Strep-Sip3∆351-469 could be detected
using the fluorogenic substrate, Z-Phe-Arg-AMC (Fig. 23) which indicated that Sip3∆351-469
produced from transient expression in N. benthamiana could be used in the following
biochemical assays.
2.4 The C-terminus of Stp1 inhibits the activity of the maize cysteine
protease, Sip3
Stp1 is secreted into apoplast during biotrophic growth (Schipper, 2009) which suggests
that Stp1 may influence the activity of the secreted maize cysteine protease, Sip3. Sip3
(Sip3∆351-469) was incubated with either Stp1∆136-432 or Stp1433-515 and the protease activity
was determined. E64, an irreversible inhibitor was used as positive control and Aro7 was
used as negative control. Stp1∆136-432, Stp1433-515 and E64 inhibited the activity of Sip3
while Aro7 showed little influence on the activity of Sip3 (Fig. 23). This suggested that the
cysteine protease inhibitor function of Stp1 could reside in the C-terminal domain of Stp1.
However, the possibility that the N-terminal domain of Stp1 may contribute to cysteine
protease inhibition could not be ruled out from this assay.
To rule out the possibility that the inhibition of Sip3 was caused by substrate inhibition,
Stp1∆136-432, Stp1433-515 and Aro7 were visualized through SDS-PAGE after incubation with
Sip3 using respective proteins without incubation with Sip3 as control. After 0.5 hour
incubation, none of Stp1∆136-432, Stp1433-515 and Aro7 showed obvious degradation (Fig. 24).
After 24 hours incubation, no obvious degradation was observed for Stp1∆136-432 and Aro7,
but Stp1433-515 was heavily degraded (Fig. 24). However, the degradation of the control
protein (Stp1433-515 without incubation with Sip3) was even heavier than Stp1433-515
incubated with Sip3 (Fig. 24). This suggested that Stp1433-515 was not degraded by Sip3 and
Results
35
Sip3 might stabilize Stp1433-515. These results indicated that neither Stp1∆136-432 nor Stp1433-
515 was the substrate of Sip3. In addition, without incubation with Sip3, Stp1433-515 was
heavily degraded while Stp1∆136-432 was stable after 24 hours (Fig. 24). This suggests that
N-terminus of Stp1 may be essential for the stability of C-terminus of Stp1.
2.5 Localization of Stp1 in infected plants
Transient expression through biolistic bombardment showed that Stp1 as well as C-terminal
domain of Stp1 were localized in the nucleus of maize cells (Schipper, 2009). In addition,
Fig. 23. Stp1 inhibits the activity of Sip3. 10 µM of cysteine protease inhibitor either proteins or E64 was used in the assay. The result was average of three repeats.
Fig. 24. None of Stp1∆136-432, Stp1433-515 and Aro7 was degraded by Sip3. Upper panel, SDS visualization of Stp1∆136-432, Stp1433-515 and Aro7 which were incubated with Sip3 for 0.5 hour. Lower panel, SDS visualization of Stp1∆136-432, Stp1433-515 and Aro7 which were incubated with Sip3 for 24 hours. Lane 1, His-Aro7 without incubation with Sip3; Lane 2, His-Stp1∆136-432 without incubation with Sip3; Lane 3, His-Stp1433-515 without incubation with Sip3; Lane 4, His-Aro7 incubated with Strep-Sip3; Lane 5, His-Stp1∆136-432 incubated with Strep-Sip3; Lane 6, His-Stp1433-515 incubated with Strep-Sip3; Pageruler, unstained Low Range Protein Ladder (Fisher Scientific). The red arrows showed His-Aro7 (36.1 kDa), His-Stp1∆136-432 (24.4 kDa) and His-Stp1433-515 (12.5 kDa). The red cycles denote His-Stp1433-515.
Results
36
Stp1 was secreted into apoplast during biotrophic growth (Schipper, 2009). However,
translocation of Stp1 from fungal hyphae into plant cells was never visualized. To find out
whether Stp1 can be translocated inside plant cells, a nuclear targeting assay using nuclear
localization signal (NLS) was performed (Khang et al., 2010). For this I constructed an U.
maydis strain in which Stp1 was fused with mCherry and NLS and its expression was
driven by the strong in planta induced cmu1 promoter (Djamei et al., 2011). The plasmid
p123pcmu-stp1-mcherry-HA-NLS was constructed and inserted into the ip locus of
SG200∆stp1 to produce SG200∆stp1-stp1-mcherry-HA-NLS. As negative control, I
constructed a strain SG200pcmu1-mcherry-HA-NLS expressing mCherry-HA-NLS under
the cmu1 promoter fused to the signal peptide of Stp1. Fluorescent signal of Stp1 fusion
protein could be detected inside the plant nucleus (Fig. 25). However, the signal was very
weak and could only be detected using high laser power.
To substantiate the translocation of Stp1, SG200∆stp1-stp1-mcherry-HA-NLS infected
maize leaves were stained using immunocytochemical techniques described by Sauer et al.
(2006). As first antibody, a monoclonal anti-HA antibody produced in mouse (Sigma) was
used, the secondary antibody was rabbit anti-mouse IgG (Life Technologies) and as third
antibody, Cy3 conjugated goat anti-rabbit IgG (Millipore) was used. As negative controls,
Fig. 25. Stp1-mCherry-HA-NLS may be translocated into plant cells. The maize leaves infected with SG200∆stp1-stp1-mcherry-HA-NLS and SG200pcmu1-mcherry-HA-NLS were taken three days after infection. The phenotype was observed using confocal microscopy. The figures are the overlay of bright field and mCherry channel. The green arrows show Stp1 fusion protein inside nuclei.
Results
37
SG200 was used to test the background of the staining method and SG200pcmu1-mcherry-
HA-NLS was used as internal control. During confocal microscopy observation, beside
DAPI channel for nuclear stain, Alexa fluor 633 channel was also observed as a control
channel to detect autofluorescence. In SG200∆stp1-stp1-mcherry-HA-NLS infected maize
leaves, fluorescent signals were observed in the nucleus as well as cytosol of the plant cell
close to the hyphal tip where the secretion of effectors was robust (Fig. 26). Additionally,
these patchy signals of Stp1-mCherry-HA-NLS are different from the evenly distributed
autofluorescence of SG200 (Fig. 26). In SG200pcmu1-mcherry-HA-NLS infected maize
leaves, fluorescent signals could also be detected in the nuclei of the plant cells. However,
fungal hyphae were never observed adjacent to these nuclei. Additionally, the fluorescent
signals are so dispersed that many nuclei in this experiment are producing fluorescent
signals (Fig. 26), which reflects unspecific signals.
2.6 Differential expression analysis of U. maydis infected plants by RNA-Seq
Previous experiments had suggested that the N- and C- terminal domains of Stp1 could
have distinct functions. To determine if stp1 mutants expressing either N- or C- terminus of
stp1 could trigger distinct plant responses, RNA-Seq was performed to detect differentially
expressed genes of U. maydis infected maize leaves.
2.6.1 Sequencing and mapping of reads to the maize genome
Plant samples infected with U. maydis (SG200, SG200∆stp1, SG200∆stp1-stp1∆40-136 and
SG200∆stp1-stp1∆432-515) were collected at 12 hpi and 24 hpi. For each sample, three
independent infections were performed and plant samples were kindly collected by Karin
Münch. After extraction of total RNA and depletion of rRNA, samples were subjected to
Illumina sequencing by the Max-Planck Genome-Centre-Cologne (Cologne, Germany). A
436.8 million 96-base reads RNA-Seq dataset was generated from Illumina sequencing
(Table. 3). The sequencing data was then mapped to the genomic sequence of maize which
consists of 39,656 genes (Filtered gene set of the release 5b (http://ftp.maizesequence.org))
using Clc genomics workbench 4. 373.0 million reads (85.4% of the total reads) could be
mapped to the reference and 63.8 million reads (14.6% of the total reads) containing the U.
maydis transcriptome could not be mapped to the reference (Table. 3). Specifically,
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26,944 to 34,268 (77.2% to 86.4% of all the genes in maize genome) distinct genes with at
least one read were identified from individual samples (Table. 3).
2.6.2 Strategy for detection of differentially expressed genes
The Clc genomics workbench adopted the reads per kilobase per million mapped reads
(RPKM) metric which normalizes a transcript read count by RNA length and total read
number (Mortazavi et al., 2008). This metric for normalization is too simple and may give
rise to higher false positive rates and lower power to detect true expression differences
(Robinson & Oshlack, 2010). Robinson and Smyth’s EdgeR (R/Bioconductor packages,
http://bioconductor.org), which is first proposed method based on negative binomial (NB)
model, employs an exact test for the NB distribution based on the trimmed mean of M
values (TMM) normalized data (Kvam et al., 2012, Gao et al., 2010). Hardcastle and
Kelly’s baySeq (R/Bioconductor packages, http://bioconductor.org) employs an empirical
Bayesian analysis approach to detect differential expression (Hardcastle & Kelly, 2010).
Multiple sample groups (more than two groups) comparison function was implemented in
both methods, but this function has not yet been tested experimentally. In simulation tests,
the false discovery rate of multiple sample groups comparison was considerably higher than
pair-wise comparison (Hardcastle & Kelly, 2010, Robinson et al., 2010). Both EdgeR and
baySeq were used for pair-wise comparison of mapped genes in this study. Firstly, gene
sets of SG200∆stp1, SG200∆stp1-stp1∆40-136 and SG200∆stp1-stp1∆432-515 infected plants
were compared with gene sets of SG200 infected plants using both EdgeR and baySeq.
Only the candidates which were classified to be differentially expressed genes by both
methods were considered to be differentially expressed genes. Subsequently, the
differentially expressed gene sets were compared to discover the differences between
SG200∆stp1, SG200∆stp1-stp1∆40-136 and SG200∆stp1-stp1∆432-515. Meanwhile, gene
ontology enrichment analysis of differentially expressed gene sets was performed to
identify the enriched biological processes.
Fig. 26. Immunostaining detected Stp1 fusion protein in the nucleus. A, DAPI channel to visualize the nucleus (blue); B, Cy3 channel to visualize Stp1-mcherry-HA-NLS signal (red); C, Enlargement of the area of white rectangular in B; D, Excitation at 633 nm as a control wavelength of autofuorescence (green); E, Merging of different channels. F, Bright field channel to visualize the hyphae of SG200. The white arrows show the nuclei. The yellow arrows denote cytosolic signals of Cy3 in SG200∆stp1-stp1-mcherry-HA-NLS infected plants. The maize leaves infected with indicated U. maydis strains were taken two days after infection.
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Samples* Time Total reads Matched reads (Proportion in total
reads)
Unmatched reads (Proportion in total
reads)
Matched genes (Proportion in
total genes) SG200 R1 12 hpi 15056165 12938204 (85.9%) 2117961 (14.1%) 32583 (82.2%)
SG200 R2 12 hpi 16443430 13927677 (84.7%) 2515753 (15.3%) 32411 (81.7%)
SG200 R3 12 hpi 21140215 17725813 (83.8%) 3414402 (16.2%) 33090 (83.4%)
SG200∆stp1 R1 12 hpi 13261743 10732420 (80.9%) 2529323 (19.1%) 31476 (79.4%)
SG200∆stp1 R2 12 hpi 15778676 13633878 (86.4%) 2144798 (13.6%) 32196 (81.2%)
SG200∆stp1 R3 12 hpi 18178342 15349605 (84.4%) 2828737 (15.6%) 32552 (82.1%)
SG200∆stp1-stp1∆432-515 R1 12 hpi 17990021 15299374 (85.0%) 2690647 (15.0%) 33162 (83.6%)
SG200∆stp1-stp1∆432-515 R2 12 hpi 21330941 18021481 (84.5%) 3309460 (15.5%) 32620 (82.3%)
SG200∆stp1-stp1∆432-515 R3 12 hpi 20723065 17733613 (85.6%) 2989452 (14.4%) 33243 (83.8%)
SG200∆stp1-stp1∆40-136 R1 12 hpi 13037991 11311317 (86.8%) 1726674 (13.2%) 30619 (77.2%)
SG200∆stp1-stp1∆40-136 R2 12 hpi 15731348 13588764 (86.4%) 2142584 (13.6%) 31058 (78.3%)
SG200∆stp1-stp1∆40-136 R3 12 hpi 15676527 13672988 (87.2%) 2003539 (12.8%) 30346 (76.5%)
SG200 R1 24 hpi 17015081 14689755 (86.3%) 2325326 (13.7%) 31364 (79.1%)
SG200 R2 24 hpi 18827855 16527516 (87.8%) 2300339 (12.2%) 30202 (76.2%)
SG200 R3 24 hpi 20097898 17836964 (88.8%) 2260934 (11.2%) 30636 (77.3%)
SG200∆stp1 R1 24 hpi 20601786 17462086 (84.8%) 3139700 (15.2%) 32444 (81.8%)
SG200∆stp1 R2 24 hpi 18050529 15678870 (86.9%) 2371659 (13.1%) 31362 (79.1%)
SG200∆stp1 R3 24 hpi 23010721 19407234 (84.3%) 3603487 (15.7%) 32769 (82.6%)
SG200∆stp1-stp1∆432-515 R1 24 hpi 14028247 11841911 (84.4%) 2186336 (15.6%) 32170 (81.1%)
SG200∆stp1-stp1∆432-515 R2 24 hpi 15829361 13602005 (85.9%) 2227356 (14.1%) 33099 (83.5%)
SG200∆stp1-stp1∆432-515 R3 24 hpi 24880482 21017876 (84.5%) 3862606 (15.5%) 34268 (86.4%)
SG200∆stp1-stp1∆40-136 R1 24 hpi 16879123 14377147 (85.2%) 2501976 (14.8%) 33002 (83.2%)
SG200∆stp1-stp1∆40-136 R2 24 hpi 23423161 19885792 (84.9%) 3537369 (15.1%) 33381 (84.2%)
SG200∆stp1-stp1∆40-136 R3 24 hpi 19848841 16752063 (84.4%) 3096778 (15.6%) 34041 (85.8%)
Total 436841549 373014353 (85.4%) 63827196 (14.6%)
2.6.3 SG200∆stp1-stp1∆40-136 triggered distinct plant responses from SG200∆stp1-
stp1∆432-515
At 24 hpi, compared to SG200 infected plants, 130 genes were up-regulated and 52 genes
were down-regulated in SG200∆stp1 infected plants while 78 genes were up-regulated and
48 genes were down-regulated in SG200∆stp1-stp1∆432-515 infected plants and 210 genes
were up-regulated and 52 genes were down-regulated in SG200∆stp1-stp1∆40-136 infected
plants. At 12 hpi, the difference between SG200 and stp1 mutants was not as evident as for
the 24 hpi time point (data not shown). Therefore, the following analysis focused on the
dataset from 24 hpi. Clustering of all the differentially expressed genes in plant samples
*, The samples are maize plants infected with indicated U. maydis strains. R1, R2 and R3 indicate three biological replicates, corresponding to three independent infections.
Table 3. Summary of samples mapped to reference
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infected with SG200∆stp1, SG200∆stp1-stp1∆432-515 and SG200∆stp1-stp1∆40-136 by
transcript abundance showed that SG200∆stp1-stp1∆40-136 triggered distinct plant responses
from SG200∆stp1 and SG200∆stp1-stp1∆432-515 (Fig. 27).
Comparison of differentially expressed gene sets of stp1 mutants showed that there were 58
up-regulated genes and 30 down-regulated genes shared by plants infected with
SG200∆stp1, SG200∆stp1-stp1∆432-515 and SG200∆stp1-stp1∆40-136 (Fig. 28 and Table. 4).
Gene ontology enrichment analysis revealed that up-regulated genes are enriched in
biological processes of cell wall organization or biogenesis, oxidation-reduction, protein
modification, phosphorus metabolism, defense response to bacteria and fungi, respiratory
gas exchange and nitrogen compound transport (See Supplementary Table. 2). Down-
regulated genes are enriched in organic substance metabolism, small molecular metabolism,
cell wall macromolecular metabolism, response to wounding and negative regulation of
biosynthetic and macromolecular metabolism, etc. (See Supplementary Table. 2). The
enrichment of cell wall organization or biogenesis process and protein modification process
indicated the onset of a broad metabolic reprogramming of in infected tissue (Doehlemann
et al., 2008). Interestingly, response to wounding is down-regulated in all stp1 mutants.
During infection, SG200 proliferates profusely and may cause more damage to plants than
SG200∆stp1, SG200∆stp1-stp1∆432-515 and SG200∆stp1-stp1∆40-136. Therefore, stp1 mutants
induced less plant responses to wounding than SG200.
Compared to SG200∆stp1 and SG200∆stp1-stp1∆432-515, there are 95 up-regulated genes
and 6 down-regulated genes only identified in SG200∆stp1-stp1∆40-136 infected plants (Fig.
28 and Table. 5). Gene ontology enrichment analysis demonstrated that up-regulated genes
are enriched in biological processes of phosphorus metabolism, cell wall organization or
biogenesis, protein modification, cellular lipid metabolism, cell death and defense response
to bacteria and fungi (See Supplementary Table. 2). Down-regulated genes have no
enriched biological processes. There are 2 up-regulated genes and 8 down-regulated genes
only identified in SG200∆stp1-stp1∆432-515 infected plants (Fig. 28 and Table. 6). Gene
ontology enrichment analysis revealed that up-regulated genes have no enriched biological
processes while down-regulated genes are enriched in cell wall organization or biogenesis
process (See Supplementary Table. 2) which indicated metabolic reprogramming in
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Fig. 27. SG200∆stp1-stp1∆40-136
triggered distinct plant responses from SG200∆stp1 and SG200∆stp1-stp1∆432-515. The abundance of genes from low to high is shown in colors from green to red. Different classes of maize genes differentially expressed in plants infected with indicated U. maydis strains are shown by the brace on the right.
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infected tissue (Doehlemann et al., 2008). This process is also enriched in both up and
down-regulated genes common to plants infected with SG200∆stp1, SG200∆stp1-stp1∆432-
515 and SG200∆stp1-stp1∆40-136. There are 15 up-regulated genes and 12 down-regulated
genes only identified in SG200∆stp1 infected plants (Fig. 28 and Table. 7). Gene ontology
enrichment analysis showed that up-regulated genes are enriched in cell wall organization
or biogenesis and phosphorus metabolic processes while down-regulated genes are enriched
in negative regulation of primary metabolic, biosynthetic, and macromolecular metabolic
processes (Table. 7)
There are 9 up-regulated genes and 2 down-regulated genes shared by SG200∆stp1 and
SG200∆stp1-stp1∆432-515 infected plants (Fig. 28 and Table. 8). Gene ontology enrichment
analysis demonstrated that phosphorus metabolism, cell wall organization or biogenesis and
protein modification processes are enriched in up-regulated genes while there are no
biological processes enriched in down-regulated genes (See Supplementary Table. 2).
There are 48 up-regulated genes and 8 down-regulated genes shared by SG200∆stp1 and
SG200∆stp1-stp1∆40-136 infected plants (Fig. 28 and Table. 9). Gene ontology enrichment
analysis showed that up-regulated genes are enriched in phosphorus metabolism, protein
modification, gamma-aminobutyric acid signaling pathway and anion transport processes
Fig. 28. Comparison of differentially expressed gene sets of plants infected with stp1 mutants. The numbers in different areas refer to the numbers of differentially expressed genes of indicated strains.
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while there is no biological process enriched in the down-regulated genes (See
Supplementary Table. 2). There are 9 up-regulated genes and 8 down-regulated genes
shared by SG200∆stp1-stp1∆432-515 and SG200∆stp1-stp1∆40-136 infected plants (Fig. 28 and
Table. 10). Gene ontology enrichment analysis revealed that up-regulated genes are
enriched in phosphorus metabolic process while down-regulated genes are enriched in
indole-containing compound metabolic process (See Supplementary Table. 2).
Gene ID GO descriptions
AC208897.3_FG004*
GRMZM2G003970
GRMZM2G004519 protein binding|zinc ion binding
GRMZM2G022699
GRMZM2G026143
GRMZM2G034611 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G036564
GRMZM2G043857
GRMZM2G056329 ATP binding
GRMZM2G063880 transcription factor activity|sequence-specific DNA binding|regulation of transcription
GRMZM2G065585 hydrolase activity, hydrolyzing O-glycosyl compounds|carbohydrate metabolic process|membrane
GRMZM2G067591 monooxygenase activity|iron ion binding|electron carrier activity|heme binding|oxidation reduction
GRMZM2G072406
GRMZM2G074611
GRMZM2G074743 mitochondrial envelope|respiratory gaseous exchange|oxidation reduction
GRMZM2G075884 protein kinase activity|protein serine/threonine kinase activity|binding|ATP binding|sugar binding|protein aminoacid phosphorylation
GRMZM2G077914 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G079491
GRMZM2G089506 aspartic-type endopeptidase activity|proteolysis
GRMZM2G099454 chitinase activity|carbohydrate metabolic process|chitin catabolic process|cell wall macromolecule catabolicprocess
GRMZM2G099467 iron ion binding|oxidoreductase activity|oxidation reduction
GRMZM2G113421 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G116629
GRMZM2G117989 homoiothermy|defense response to bacterium|ice binding|response to freezing|defense response to fungus
GRMZM2G118800 aldehyde dehydrogenase [NAD(P)+] activity|cellular aldehyde metabolic process|metabolicprocess|oxidoreductase activity|oxidation reduction
GRMZM2G118809 monooxygenase activity|iron ion binding|electron carrier activity|heme binding|oxidation reduction
GRMZM2G119755
GRMZM2G123107 hydrolase activity, hydrolyzing O-glycosyl compounds|carbohydrate metabolic process
GRMZM2G124175 oxidoreductase activity|oxidation reduction
GRMZM2G131099
GRMZM2G135165 structural constituent of ribosome|intracellular|ribosome|translation|homoiothermy|ice binding|response to freezing
GRMZM2G145045 protein kinase activity|protein serine/threonine kinase activity|calcium ion binding|ATP binding|protein aminoacid phosphorylation
GRMZM2G147752 monooxygenase activity|iron ion binding|electron carrier activity|heme binding|oxidation reduction
Table 4. Differentially expressed maize genes common to SG200∆stp1, SG200∆stp1-stp1∆432-515 and
SG200∆stp1-stp1∆40-136 infected plants
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GRMZM2G153208 lipid metabolic process|phosphatidylglycerophosphatase activity|nutrient reservoir activity
GRMZM2G154870 monooxygenase activity|iron ion binding|electron carrier activity|heme binding|oxidation reduction
GRMZM2G160614 receptor activity|transporter activity|ion channel activity|extracellular-glutamate-gated ion channelactivity|transport|ion transport|membrane|integral to membrane|substrate-specific transmembrane transporteractivity|transmembrane transport
GRMZM2G160710 metal ion transport|metal ion binding
GRMZM2G161521
GRMZM2G174449
GRMZM2G176206 protein kinase activity|protein serine/threonine kinase activity|protein binding|ATP binding|protein amino acidphosphorylation
GRMZM2G176472
GRMZM2G178645 nucleotide binding|ATP binding|nucleoside-triphosphatase activity
GRMZM2G180659 amino acid transport|membrane
GRMZM2G315726 integral to membrane
GRMZM2G337594
GRMZM2G358153 hydrolase activity, hydrolyzing O-glycosyl compounds|chitinase activity|carbohydrate metabolic process|chitincatabolic process
GRMZM2G374309 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G402631
GRMZM2G415529 nucleotide binding|ATP binding|membrane|ATPase activity|nucleoside-triphosphatase activity
GRMZM2G423202
GRMZM2G426917 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G431039 hydrolase activity, hydrolyzing O-glycosyl compounds|carbohydrate metabolic process
GRMZM2G436448 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G453805 hydrolase activity, hydrolyzing O-glycosyl compounds|chitinase activity|carbohydrate metabolic process|chitincatabolic process
GRMZM2G465226 extracellular region
GRMZM2G475948 protein kinase activity|protein serine/threonine kinase activity|binding|ATP binding|protein amino acidphosphorylation
GRMZM5G868679 acid phosphatase activity|hydrolase activity|metal ion binding
GRMZM5G899851 catalytic activity|protein kinase activity|ATP binding|protein amino acid phosphorylation|electron carrieractivity|oxidoreductase activity|metal ion binding|FAD binding|iron-sulfur cluster binding|oxidation reduction
AC196110.4_FG004
AC206201.3_FG004
GRMZM2G000326 serine-type endopeptidase inhibitor activity|response to wounding
GRMZM2G002178 monooxygenase activity|iron ion binding|electron carrier activity|heme binding
GRMZM2G005954
GRMZM2G009232 monooxygenase activity|iron ion binding|electron carrier activity|heme binding|oxidation reduction
GRMZM2G039993 methyltransferase activity
GRMZM2G042789 serine-type endopeptidase inhibitor activity|response to wounding
GRMZM2G042895 nucleus|transcription regulator activity|regulation of transcription
GRMZM2G043336 carbon fixation|ribulose-bisphosphate carboxylase activity
GRMZM2G049211
GRMZM2G049538 magnesium ion binding|metabolic process|lyase activity
GRMZM2G064360 chitinase activity|chitin catabolic process|chitin binding|cell wall macromolecule catabolic process
GRMZM2G094304 glutamine metabolic process|hydrolase activity
GRMZM2G096680 serine-type endopeptidase inhibitor activity|response to wounding
GRMZM2G103197 electron carrier activity|oxidoreductase activity
GRMZM2G108514 cellular amino acid and derivative metabolic process|carboxy-lyase activity|carboxylic acid metabolicprocess|pyridoxal phosphate binding
GRMZM2G119705 neuropeptide Y receptor activity|G-protein coupled receptor protein signaling pathway|integral tomembrane|negative regulation of translation|rRNA N-glycosylase activity
GRMZM2G127336 magnesium ion binding|metabolic process|lyase activity
GRMZM2G159179 monooxygenase activity|iron ion binding|electron carrier activity|heme binding|oxidation reduction
GRMZM2G167698 monooxygenase activity|iron ion binding|electron carrier activity|heme binding|oxidation reduction
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GRMZM2G174562 serine-type endopeptidase inhibitor activity|extracellular region
GRMZM2G312061 cysteine-type endopeptidase inhibitor activity
GRMZM2G329029 monooxygenase activity|iron ion binding|electron carrier activity|heme binding|oxidation reduction
GRMZM2G338160
GRMZM2G353444 triglyceride lipase activity|lipid metabolic process
GRMZM2G359581 cysteine-type endopeptidase inhibitor activity
GRMZM2G565911
GRMZM5G833406 lysosphingolipid and lysophosphatidic acid receptor activity|protein binding|G-protein coupled receptor proteinsignaling pathway|metabolic process|integral to membrane|hydrolase activity
GRMZM5G847669 serine-type endopeptidase inhibitor activity|extracellular region
Gene ID GO descriptions
AC204515.4_FG006*
AC214360.3_FG001
AC225176.2_FG003
GRMZM2G001332
GRMZM2G005633 chitinase activity|carbohydrate metabolic process|chitin catabolic process|chitin binding|metabolic process|oxidoreductase activity|cell wall macromolecule catabolic process|oxidation reduction
GRMZM2G006894 catalytic activity|ATP binding|ATP biosynthetic process|cation transport|metabolic process|ATPase activity, coupled to transmembrane movement of ions, phosphorylative mechanism|membrane|integral to membrane|hydrolase activity, acting on acid anhydrides, catalyzing transmembrane movement of substances|ATPase activity
GRMZM2G009045 binding|transport|membrane|transmembrane transport
GRMZM2G014071 triglyceride lipase activity|lipid metabolic process
GRMZM2G014395 monooxygenase activity|iron ion binding|electron carrier activity|heme binding|oxidation reduction
GRMZM2G017164 protein kinase activity|protein serine/threonine kinase activity|ATP binding|sugar binding|protein amino acid phosphorylation
GRMZM2G018707
GRMZM2G020508
GRMZM2G021378 SNAP receptor activity|protein binding|intracellular protein transport|membrane|vesicle-mediated transport
GRMZM2G022972
GRMZM2G028306 magnesium ion binding|metabolic process|lyase activity
GRMZM2G028713 nucleotide binding|protein binding|ATP binding|apoptosis|defense response|nucleoside-triphosphatase activity
GRMZM2G032602 nucleotide binding|protein binding|ATP binding|apoptosis|defense response|nucleoside-triphosphatase activity
GRMZM2G037209
GRMZM2G039362
GRMZM2G039639
GRMZM2G044481 magnesium ion binding|metabolic process|lyase activity
GRMZM2G051921 chitinase activity|carbohydrate metabolic process|chitin catabolic process|chitin binding|cell wall macromolecule catabolic process
GRMZM2G052266 oxidoreductase activity
GRMZM2G059496
GRMZM2G059740 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G064603 nucleotide binding|ATP binding|membrane|phosphotransferase activity, alcohol group as acceptor|ATPase activity|nucleoside-triphosphatase activity|homoiothermy|ice binding|response to freezing
GRMZM2G065655 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G069335
GRMZM2G071436
GRMZM2G072529 iron ion binding|oxidoreductase activity|oxidation reduction
Table 5. Differentially expressed maize genes identified only in SG200∆stp1-stp1∆40-136 infected plants
*: The red font denotes up regulated genes. The black font denotes down regulated genes. See Supplementary Table. 1 for GO terms corresponding to the indicated genes. Gene ID: MaizeGDB, (Harris et al., 2005)
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GRMZM2G076394
GRMZM2G080103
GRMZM2G081127 DNA binding|nucleus
GRMZM2G082199
GRMZM2G085974
GRMZM2G086869 catalytic activity|metabolic process|hydrolase activity
GRMZM2G088819 calcium ion binding
GRMZM2G097706 metabolic process|oxidoreductase activity|oxidation reduction
GRMZM2G100475
GRMZM2G101405 transcription factor activity|sequence-specific DNA binding|regulation of transcription
GRMZM2G106177 integral to membrane
GRMZM2G109056 iron ion binding|lipoxygenase activity|oxidoreductase activity, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen|metal ion binding|oxidation reduction
GRMZM2G113512 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G117942 defense response to bacterium|defense response to fungus
GRMZM2G117971 defense response to bacterium|defense response to fungus
GRMZM2G123119 DNA binding|transcription factor activity|regulation of transcription, DNA-dependent
GRMZM2G125762 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G125775 zinc ion binding
GRMZM2G126261 peroxidase activity|response to oxidative stress|heme binding|nutrient reservoir activity|oxidation reduction
GRMZM2G129189 chitinase activity|carbohydrate metabolic process|chitin catabolic process|chitin binding|cell wall macromolecule catabolic process
GRMZM2G129860 monooxygenase activity|iron ion binding|electron carrier activity|heme binding|oxidation reduction
GRMZM2G133430
GRMZM2G135385 heme binding
GRMZM2G148087 transcription factor activity|sequence-specific DNA binding|regulation of transcription
GRMZM2G148904 metabolic process|methyltransferase activity
GRMZM2G151204 ubiquitin ligase complex|ubiquitin-protein ligase activity|binding|protein ubiquitination
GRMZM2G152739 triglyceride lipase activity|lipid metabolic process|oxidoreductase activity
GRMZM2G157218
GRMZM2G158045 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G160739 nutrient reservoir activity
GRMZM2G162829 nucleic acid binding|endonuclease activity|protein kinase activity|protein serine/threonine kinase activity|protein binding|ATP binding|extrachromosomal circular DNA|protein amino acid phosphorylation|pathogenesis
GRMZM2G163054 transcription factor activity|sequence-specific DNA binding|regulation of transcription
GRMZM2G163307
GRMZM2G164640
GRMZM2G171400
GRMZM2G173536
GRMZM2G177883 protein kinase activity|protein serine/threonine kinase activity|protein binding|ATP binding|protein amino acid phosphorylation
GRMZM2G180080 nucleotide binding|ATP binding|nucleoside-triphosphatase activity
GRMZM2G181227 lysozyme activity|catalytic activity|metabolic process|cell wall macromolecule catabolic process
GRMZM2G314396 protein kinase activity|protein serine/threonine kinase activity|calcium ion binding|ATP binding|protein amino acid phosphorylation
GRMZM2G329002
GRMZM2G333448 intracellular signaling pathway
GRMZM2G334181
GRMZM2G334336 metabolic process|transferase activity, transferring hexosyl groups
GRMZM2G341499 G-protein coupled receptor activity|GABA-B receptor activity|ionotropic glutamate receptor activity|transporter activity|extracellular-glutamate-gated ion channel activity|transport|G-protein coupled receptor protein signaling pathway|membrane|integral to membrane|outer membrane-bounded periplasmic space
GRMZM2G372068 metabolic process|transferase activity, transferring hexosyl groups
GRMZM2G374827 catalytic activity|glucosylceramidase activity|sphingolipid metabolic process|glucosylceramide catabolic process|membrane|integral to membrane|homoiothermy|ice binding|response to freezing
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GRMZM2G400470 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G410991 cell wall macromolecule catabolic process
GRMZM2G414252 nucleus|transcription regulator activity|regulation of transcription
GRMZM2G416632 glutathione transferase activity|cytoplasm|metabolic process
GRMZM2G433076 protein kinase activity|protein serine/threonine kinase activity|calcium ion binding|ATP binding|protein amino acid phosphorylation
GRMZM2G434363 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G439578 hydrolase activity|hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds
GRMZM2G443843 protein kinase activity|protein serine/threonine kinase activity|binding|ATP binding|protein amino acid phosphorylation
GRMZM2G452121 protein kinase activity|protein serine/threonine kinase activity|binding|ATP binding|protein amino acid phosphorylation
GRMZM2G459663 calcium ion binding|homoiothermy|ice binding|response to freezing
GRMZM2G464157 hydrolase activity
GRMZM2G474546 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G493395 catalytic activity|metabolic process|1-deoxy-D-xylulose-5-phosphate synthase activity|terpenoid biosynthetic process
GRMZM5G838907
GRMZM5G841893 monooxygenase activity|potassium ion transport|metabolic process|cation transmembrane transporter activity|oxidoreductase activity|oxidation reduction
GRMZM5G849600 galanin receptor activity|nucleus|G-protein coupled receptor protein signaling pathway|integral to membrane|transcription regulator activity|regulation of transcription
GRMZM5G863420 transcription factor activity|homoiothermy|sequence-specific DNA binding|regulation of transcription|ice binding|response to freezing
GRMZM5G892675 nucleotide binding|ATP binding|membrane|ATPase activity|nucleoside-triphosphatase activity
GRMZM2G012160 cysteine-type endopeptidase inhibitor activity
GRMZM2G075456
GRMZM2G109252
GRMZM2G111954 oxidoreductase activity|oxidation reduction
GRMZM2G470882
GRMZM5G844309
Gene ID GO descriptions
GRMZM2G011160*
GRMZM2G422045 inositol or phosphatidylinositol phosphatase activity
GRMZM2G059693 triglyceride lipase activity|lipid metabolic process
GRMZM2G097297 methyltransferase activity|O-methyltransferase activity|protein dimerization activity
GRMZM2G109130 iron ion binding|lipoxygenase activity|oxidoreductase activity, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen|metal ion binding|oxidation reduction
GRMZM2G161905
GRMZM2G165192 transferase activity, transferring acyl groups other than amino-acyl groups
GRMZM2G320117
GRMZM2G389582 chitinase activity|chitin catabolic process|cell wall macromolecule catabolic process
GRMZM2G436020
Table 6. Differentially expressed maize genes identified only in SG200∆stp1-stp1∆432-515 infected plants
*: The red font denotes up regulated genes. The black font denotes down regulated genes. See Supplementary Table. 1 for GO terms corresponding to the indicated genes. Gene ID: MaizeGDB, (Harris et al., 2005)
*: The red font denotes up regulated genes. The black font denotes down regulated genes. See Supplementary Table. 1 for GO terms corresponding to the indicated genes. Gene ID: MaizeGDB, (Harris et al., 2005)
Results
49
Gene ID GO descriptions
AC231745.1_FG003*
GRMZM2G047791
GRMZM2G056875 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G062974 chitinase activity|carbohydrate metabolic process|chitin catabolic process|chitin binding|cell wall macromolecule catabolic process
GRMZM2G092718 chromatin|DNA binding|nucleus|metal ion transport|metal ion binding
GRMZM2G096090
GRMZM2G098102 cysteine-type endopeptidase activity|proteolysis|cysteine-type peptidase activity
GRMZM2G120016 metabolic process|transferase activity, transferring hexosyl groups
GRMZM2G136372
GRMZM2G145461 chitinase activity|chitin catabolic process|chitin binding|cell wall macromolecule catabolic process
GRMZM2G154828 monooxygenase activity|iron ion binding|electron carrier activity|heme binding|oxidation reduction
GRMZM2G163494
GRMZM2G313104 homoiothermy|ice binding|response to freezing
GRMZM2G427815 peroxidase activity|response to oxidative stress|heme binding|oxidation reduction
GRMZM2G449817 protein kinase activity|protein serine/threonine kinase activity|protein binding|ATP binding|protein amino acid phosphorylation
GRMZM2G006937 catalytic activity|intramolecular transferase activity
GRMZM2G047713 negative regulation of translation|rRNA N-glycosylase activity
GRMZM2G069736
GRMZM2G071023 NAD+ kinase activity|metabolic process
GRMZM2G156861 iron ion binding|lipoxygenase activity|oxidoreductase activity, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen|metal ion binding|oxidation reduction
GRMZM2G158316
GRMZM2G160990 dopamine receptor activity|G-protein coupled receptor protein signaling pathway|integral to membrane
GRMZM2G173596
GRMZM2G173809 negative regulation of translation|rRNA N-glycosylase activity
GRMZM2G337387
GRMZM2G528190
GRMZM5G815098 serine-type endopeptidase inhibitor activity|extracellular region
Gene ID GO descriptions
GRMZM2G023827*
GRMZM2G024024 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acidphosphorylation|cell wall macromolecule catabolic process
GRMZM2G059214 protein kinase activity|protein serine/threonine kinase activity|protein binding|ATP binding|protein amino acidphosphorylation
GRMZM2G095126 hydrolase activity, hydrolyzing O-glycosyl compounds|protein binding|carbohydrate metabolic process|dicarboxylic acid transport|membrane|sodium:dicarboxylate symporter activity
GRMZM2G126975 extracellular region
GRMZM2G159908 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G177991
GRMZM2G178753 protein kinase activity|protein serine/threonine kinase activity|protein binding|ATP binding|protein amino acidphosphorylation|phosphopantetheine binding
Table 7. Differentially expressed maize genes identified only in SG200∆stp1 infected plants
Table 8. Differentially expressed maize genes common to SG200∆stp1 and SG200∆stp1-stp1∆432-515
infected plants
*: The red font denotes up regulated genes. The black font denotes down regulated genes. See Supplementary Table. 1 for GO terms corresponding to the indicated genes. Gene ID: MaizeGDB, (Harris et al., 2005)
Results
50
GRMZM2G316474 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G038153 magnesium ion binding|metabolic process|lyase activity
GRMZM2G371793 acid phosphatase activity
Gene ID GO descriptions
AC205012.3_FG002*
AC214817.3_FG007
GRMZM2G007477 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G026189 GTPase activity|ATP binding|GTP binding|apoptosis|protein complex|protein polymerization
GRMZM2G026203 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G032910 catalytic activity|aminopeptidase activity|proteolysis
GRMZM2G041356 catalytic activity|carbohydrate metabolic process|isomerase activity|carbohydrate binding
GRMZM2G050514 glutamate-ammonia ligase activity|phosphoglycerate kinase activity|glycolysis|glutamine biosynthetic process|nitrogen compound metabolic process
GRMZM2G057963 hydrolase activity
GRMZM2G061723
GRMZM2G062600 hydrolase activity, hydrolyzing O-glycosyl compounds|carbohydrate metabolic process
GRMZM2G065696
GRMZM2G077197 DNA binding|DNA topoisomerase (ATP-hydrolyzing) activity|protein binding|ATP binding|DNA topological change
GRMZM2G079219 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G079436
GRMZM2G093072 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G098346 zinc ion binding|oxidoreductase activity|oxidation reduction
GRMZM2G104125 protein kinase activity|protein serine/threonine kinase activity|calcium ion binding|ATP binding|protein amino acid phosphorylation
GRMZM2G111657 exocyst|exocytosis
GRMZM2G112222
GRMZM2G112456 transmembrane transport
GRMZM2G122654 monooxygenase activity|iron ion binding|electron carrier activity|heme binding|oxidation reduction
GRMZM2G124759 triglyceride lipase activity|lipid metabolic process
GRMZM2G126749
GRMZM2G138770 nucleotide binding|ATP binding|nucleoside-triphosphatase activity
GRMZM2G140231 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G140721 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G140752 protein kinase activity|protein serine/threonine kinase activity|GABA-A receptor activity|ATP binding|protein amino acid phosphorylation|chloride transport|gamma-aminobutyric acid signaling pathway|integral to membrane
GRMZM2G145109 protein kinase activity|protein serine/threonine kinase activity|calcium ion binding|ATP binding|protein amino acid phosphorylation
GRMZM2G146108
GRMZM2G151230
GRMZM2G165987
GRMZM2G175140 transport|ammonium transmembrane transporter activity|membrane|transmembrane transport
GRMZM2G175593 nucleotide binding|ATP binding|nucleoside-triphosphatase activity
GRMZM2G304897 protein kinase activity|protein serine/threonine kinase activity|calcium ion binding|ATP binding|sugar binding|protein amino acid phosphorylation
GRMZM2G326707 transporter activity|inorganic phosphate transmembrane transporter activity|transport|phosphate transport|integral to membrane|transmembrane transport
Table 9. Differentially expressed maize genes common to SG200∆stp1 and SG200∆stp1-stp1∆40-136
infected plants
*: The red font denotes up regulated genes. The black font denotes down regulated genes. See Supplementary Table. 1 for GO terms corresponding to the indicated genes. Gene ID: MaizeGDB, (Harris et al., 2005)
Results
51
GRMZM2G355233 protein kinase activity|protein serine/threonine kinase activity|calcium ion binding|ATP binding|protein amino acid phosphorylation
GRMZM2G361256 nucleotide binding|ATP binding|transport|integral to membrane|ATPase activity|nucleoside-triphosphatase activity|ATPase activity, coupled to transmembrane movement of substances|transmembrane transport
GRMZM2G402977
GRMZM2G431288 monooxygenase activity|iron ion binding|electron carrier activity|heme binding|oxidation reduction
GRMZM2G443829 protein kinase activity|protein serine/threonine kinase activity|binding|ATP binding|protein amino acid phosphorylation
GRMZM2G444560 nucleotide binding|ATP binding|nucleoside-triphosphatase activity
GRMZM2G452896 proteolysis|serine-type peptidase activity
GRMZM2G458095 nucleotide binding|ATP binding|nucleoside-triphosphatase activity
GRMZM2G472655 protein kinase activity|protein serine/threonine kinase activity|ATP binding|sugar binding|protein amino acid phosphorylation|G-protein coupled receptor protein signaling pathway|integral to membrane
GRMZM2G496370 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM5G879872 metal ion transport|metal ion binding
GRMZM5G897005 protein kinase activity|protein serine/threonine kinase activity|ATP binding|protein amino acid phosphorylation
GRMZM2G058149 triglyceride lipase activity|lipid metabolic process
GRMZM2G099678
GRMZM2G110567
GRMZM2G116614
GRMZM2G118610 zinc ion binding|oxidoreductase activity|oxidation reduction
GRMZM2G136453 acid phosphatase activity|hydrolase activity|metal ion binding
GRMZM2G138710
GRMZM2G496991
Gene ID GO descriptions
GRMZM2G003411* nucleotide binding|ATP binding|membrane|ATPase activity|nucleoside-triphosphatase activity
GRMZM2G055883 GTPase activity|GTP binding
GRMZM2G057116 transcription factor activity|sequence-specific DNA binding|regulation of transcription
GRMZM2G070312
GRMZM2G109830 protein kinase activity|protein serine/threonine kinase activity|protein binding|ATP binding|protein amino acidphosphorylation
GRMZM2G117454
GRMZM2G145075 protein kinase activity|protein serine/threonine kinase activity|calcium ion binding|ATP binding|protein aminoacid phosphorylation
GRMZM5G846916 hydrolase activity, hydrolyzing O-glycosyl compounds|carbohydrate metabolic process
GRMZM5G893912 nucleotide binding|ATP binding|nucleoside-triphosphatase activity
GRMZM2G015892 catalytic activity|tryptophan synthase activity|tryptophan metabolic process|metabolic process
GRMZM2G036262
GRMZM2G049695 nucleic acid binding|DNA binding|zinc ion binding|homoiothermy|ice binding|response to freezing
GRMZM2G079616 transferase activity, transferring acyl groups other than amino-acyl groups
GRMZM2G091540 protein binding|metabolic process|membrane|hydrolase activity|protein homooligomerization
GRMZM2G304474
GRMZM2G389557 DNA binding|type I hypersensitivity|regulation of transcription
GRMZM2G700208 lipid metabolic process|hydrolase activity, acting on ester bonds
Table 10. Differentially expressed maize genes common to SG200∆stp1-stp1∆432-515 and SG200∆stp1-stp1∆40-136 infected plants
*: The red font denotes up regulated genes. The black font denotes down regulated genes. See Supplementary Table. 1 for GO terms corresponding to the indicated genes. Gene ID: MaizeGDB, (Harris et al., 2005)
*: The red font denotes up regulated genes. The black font denotes down regulated genes. See Supplementary Table. 1 for GO terms corresponding to the indicated genes. Gene ID: MaizeGDB, (Harris et al., 2005)
Results
52
The differentially regulated genes identified by RNA-Seq were compared with 37 genes
involved in early defense response which were induced at 12 hpi in plants infected with
SG200 but down-regulated at 24 hpi (Doehlemann et al., 2008). Among 37 early defense
response genes, 22 were also identified by RNA-Seq analysis in plants infected with
SG200∆stp1, SG200∆stp1-stp1∆432-515 and SG200∆stp1-stp1∆40-136 (Table. 11). 9 early
defense response genes are common to plants infected with SG200∆stp1, SG200∆stp1-
stp1∆432-515 and SG200∆stp1-stp1∆40-136 (Table. 11) indicating the plant defense response
triggered by these strains. 7 genes are only identified in plants infected with SG200∆stp1-
stp1∆40-136 (Table. 11) which indicated that SG200∆stp1 expressing C-terminus of Stp1
triggered stronger plant defense response than SG200∆stp1. 6 genes are identified only in
plants infected with SG200∆stp1 or common to plants infected with SG200∆stp1 and
SG200∆stp1-stp1∆432-515 but are absent in plants infected with SG200∆stp1-stp1∆40-136
(Table. 11) which indicated that SG200∆stp1 expressing N-terminus of Stp1 induce less
plant defense response than SG200∆stp1.
Gene ID Annotations
common to SG200∆stp1, SG200∆stp1-stp1∆432-515 and
SG200∆stp1-stp1∆40-136
common to SG200∆stp1, and SG200∆stp1-stp1∆40-
136
Only in SG200∆stp1-
stp1∆40-136
Only in SG200∆stp1
GRMZM2G065585 1,3-β-glucanase √ GRMZM2G135385 Cytochrome b5 √ GRMZM2G415529 ATPase activity √ GRMZM2G145461 chitinase activity √GRMZM2G067591 Cytochrome P450 √ GRMZM2G118800 aldehyde
dehydrogenase √
GRMZM2G065696 Nuclease sbcCD
subunit √
GRMZM2G117989 Win1 √ GRMZM2G092718 late blight resistance √GRMZM2G106177 Ergosterol
biosynthetic protein √
GRMZM2G402977 Hypothetical protein √ GRMZM2G117971 Barwin like proteins √ GRMZM2G003970 hypothetical protein √ GRMZM2G109056 lipoxygenase √ GRMZM2G131099 hypothetical protein √ GRMZM2G044481 Copalyl Diphosphate
Synthase √
GRMZM2G161521 hypothetical protein √
Table 11. Differentially expressed maize genes involved in early plant defense response
Results
53
GRMZM2G026143 WIR1A protein √ GRMZM2G416632 glutathione
transferase activity √
GRMZM2G072529 acc oxidase √ GRMZM2G032910 Epoxide hydrolase 2 √ GRMZM2G136372 thaumatin-like
protein √
Gene ID: MaizeGDB, (Harris et al., 2005). See Supplementary Table. 3 for Affymetrix probe set of indicated genes.
Discussion
54
3. Discussion
This study reveals that the N- and C-terminal conserved domains of Stp1 are essential for
protein function while the variable domains are dispensable. The C-terminal domain of
Stp1 (aa 433-515) can inhibit the activity of the maize extracellular cysteine protease Sip3,
a protein identified as interactor of Stp1 in yeast two-hybrid assays. Based on the finding
that Stp1 also interacts with several cytoplasmic plant proteins it is speculated that Stp1
may be an effector with both apoplastic and cytoplasmic functions. Moreover, RNA-Seq
analysis indicates that the N- and C-terminal domains of Stp1 have distinct functions.
3.1 Domain structure of Stp1
Like most secreted effectors of U. maydis, the primary structure of Stp1 reveals no
homology to any known functional main besides a signal peptide. Dissecting the domain
structure of Stp1 is the basis for the following functional analysis.
3.1.1 The N- and C-terminal conserved domains of Stp1 are essential for protein
function while the variable domains are dispensable.
Stp1 consists of a signal peptide, a N-terminal variable domain, a N-terminal conserved
domain, a long glycine-rich variable domain in the middle part and a second conserved
domain in the C-terminus (Fig. 29). The deletion analysis conducted in this study revealed
that N- and C-terminal domains of Stp1 can be expressed separately but both are essential
for the protein function. The N-terminal variable domain is dispensable (K. Schipper,
personal communication). The glycine-rich variable domain in the middle part is also
dispensable but may have minor virulence function. In the following, the putative functions
of Stp1 as well as individual domains of it are discussed.
Fig. 29. Domain structure of Stp1. Red: signal peptide, grey: variable domains, orange: N-terminus, blue: C-terminus.
Discussion
55
3.1.2 N- and C-terminal domains of Stp1 may be essential for the stability of each other The N- and C-terminal domains of Stp1 could be separately expressed and no interaction
between these two domains was observed in Y2H assays. This raises the question why N-
and C-terminal domains of Stp1 are needed for the function of Stp1. During attempts to
purify Stp1 and separate N- and C-terminal domains, the C-terminus of Stp1 was proved to
be unstable at room temperature (Fig. 24), while the N-terminus of Stp1 was already
degraded during the purification procedure at 4 oC (Fig. 22) making it difficult to obtain
pure protein. However, Stp1∆136-432 in which N- and C-termini of Stp1 were fused was
stable during purification procedure and subsequent biochemical assays (Fig. 24). The
instability observed for the separated N- and C-terminal domains could suggest that N- and
C-terminal domains of Stp1 may be mutually essential for the stability of each other.
Mutual stabilization is not contradictory with the result that they can function when
expressed separately. During establishment of the biotrophic interaction, Stp1 appears not
to be needed in large amounts as the stp1 promoter is relatively weak compared to strong
promoters like the cmu1 promoter which is up-regulated up to hundred thousand times
compared to axenic culture during plant colonization (Schipper, 2009, Djamei et al., 2011).
Moreover, the proposed mutual stabilization may not need a stable physical interaction
which could explain why no interaction between these two domains could be detected. In
addition, the short half life of N- and C-termini of Stp1 may be already enough for their
function. However, it cannot be ruled out presently that the stability or even the interaction
between N- and C-terminal domains will be affected by glycosylation. K. Schipper has
shown that Stp1 is N-glycosylated and one N-glycosylation sites is predicted to reside in
the C-terminal conserved domain (Schipper, 2009). It will therefore be necessary to
investigate the stability and interaction after expressing these two domains in U. maydis by
IP and Co-IP respectively.
3.2 The interaction partners of Stp1
By yeast two-hybrid analysis using Stp1∆136-432 as bait twelve interaction partners could be
identified. This study complemented a study by K. Schipper (2009) who used full-length
Stp1 as bait. Interestingly, a comparison of both studies revealed that several putative
interactors, Sip10, Sip12, Sip29 and Sip31 showed interaction with full-length Stp1 but
Discussion
56
failed to interact with Stp1∆136-432. This suggests that these proteins are unspecific
interactors that contact Stp1 via the glycine-rich variable domain and allowed to discard
these from further consideration. In the following, I will first discuss the putative apoplastic
interactors of Stp1 and follow this up with a discussion of the cytoplasmic interactors.
3.2.1 The biological significance of the inhibition of Sip3 by the C-terminus of Stp1
Two apoplastic interactors, Sip1 and Sip3, were identified in this study. Sip1 is a beta-
galactosidase. There is no conceivable evidence correlating it with either plant defense
responses or the regulation of plant development and the interaction between Sip1 and Stp1
still needs to be confirmed by full-length cDNA clone. This leaves only the extracellular
papain-like cysteine protease, Sip3 which was isolated as interactor of Stp1∆136-432 but was
subsequently also shown to interact with full-length Stp1.
Cysteine proteases also referred to as thiol proteases, are comprised of 74 families
(http://merops.sanger.ac.uk) that are encoded by viruses, bacteria, protozoa, plants,
mammals and fungi (Otto & Schirmeister, 1997, Rawlings et al., 2012). Plant cysteine
proteases play an essential role in plant growth and development, senescence and
programmed cell death, signaling pathways and the response to biotic and abiotic stresses
(Leung-Toung et al., 2002).
Sip3 belongs to C1A cysteine protease subfamily (http://merops.sanger.ac.uk). Both
purified Stp1∆136-432 and Stp1433-515 could inhibit the activity of Sip3 to a level comparable
to E64, an irreversible cysteine protease inhibitor. Bioinformatic analysis demonstrated that
Stp1 had no similarity to any known cysteine protease inhibitors like aprotinin, Avr2 and
cystatin (Kunitz & Northrop, 1936, Rooney et al., 2005, Martinez et al., 2009). This
suggests that Stp1 could be a novel cysteine protease inhibitor. However, besides Stp1, the
secreted effectors Pit2 and Tin3 could also interact with the C1A class of cysteine proteases
(N. Neidig and A. Müller, personal communication). There is no conserved motif in Stp1,
Pit2 and Tin3 and mutants in these three effector genes have very distinct phenotypes (N.
Neidig, personal communication) (Schipper, 2009, Doehlemann et al., 2011) suggesting
that these three effectors have non-redundant roles. The chance that all three secreted
effectors possess distinct novel cysteine protease inhibitor motifs is very low. Therefore,
Stp1 is probably inhibiting Sip3 unspecifically rather than functioning solely as a novel
Discussion
57
cysteine protease inhibitor. Incubation of Stp1∆136-432 and Stp1433-515 with Sip3
demonstrated that Stp1 is not a substrate of Sip3 and Sip3 may stabilize the C-terminus of
Stp1 (Fig. 24). This could suggest a novel function for the interaction between Stp1 and
Sip3 that needs to be substantiated by further experiments.
During the crosstalk between hosts and pathogens, secreted effectors functioning as
cysteine protease inhibitor are playing distinct biological functions. C. fulvum secretes a
protease inhibitor Avr2 that targets the tomato cysteine protease Rcr3, an apoplastic papain-
like cysteine protease required specifically for Cf-2 mediated resistance (Song et al., 2009,
Dixon et al., 2000). P. infestans secreted effector AVRblb2 suppresses plant defense
responses by preventing the secretion of a plant papain-like protease C14 which positively
contributes to immune responses against P. infestans (Bozkurt et al., 2011). To determine
whether the C-terminus of Stp1 has the same function as Avr2 and AVRb1b2 or whether it
has a novel function, it is necessary to generate stable RNAi lines. In Su1 maize RNAi
lines, all members of this family of papain-like proteases could be silenced simultaneously.
As the generation of stable transgenic maize plants takes about 18 months, Su1 lines are not
yet available. To evaluate how important the inhibition of apoplastic cysteine protease is for
the proliferation of stp1 mutants in planta, maize plants were treated with E64 during
infection with SG200∆stp1 expressing the N-terminus of Stp1 and the colonization was
observed (data not shown). However, the colonization showed no differences with the
control in which plants were infected with SG200∆stp1 expressing N-terminus of Stp1 in
the absence of E64. This could suggest that the inhibition of cysteine proteases is either not
the main function of the C-terminus of Stp1 or that the access of E64 to cysteine proteases
is blocked by the hydrophobic surface of maize leaves and could not therefore inhibit
respective cysteine preteases.
3.2.2 The cytoplasmic maize interaction partners of Stp1 shed light on a putative
function of Stp1 in the plant cytosol
In this study, both cytoplasmic and apoplastic maize proteins were identified as interactors
of Stp1 by Y2H assays. The interactions between Stp1 and the cytoplasmic interactors need
to be analyzed biochemically particularly Sip9, Sip19, Sip16 and Sip21, which were
verified with full-length cDNA clones.
Discussion
58
Sip9 is predicted to be cell number regulator 8. CNR (cell number regulator) gene
represents a gene family. For CNR1, it was shown that it could reduce overall plant size
when ectopically overexpressed and increase organ size when its expression was
suppressed (Guo et al., 2010). It is conceivable that Stp1 could negatively regulate the
activity of CNR protein and in this way contribute to higher cell numbers observed in
infected tissue (Doehlemann et al., 2008). To test the influence of cell number regulator on
pathogenicity, it will be interesting to analyze the development of stp1 mutants in the
CNR1 silenced maize line. However, due to patent restrictions it may be difficult to obtain
Su1 lines (R. Kahmann, personal communication)
Sip19 is predicted to be serine/threonine-protein kinase. The Pto serine/threonine-protein
kinase confers resistance to bacterial speck disease through recognition of a corresponding
avirulence protein, AvrPto, from the pathogen P. syringae pv. tomato (Shan et al., 2008,
Frederick et al., 1998). To establish a connection between Sip19 and Stp1, Sip19 will be
heterologously expressed and the influence of Stp1 on the protein kinase activity of Sip19
will be tested.
Sip16, a putative CCR4-NOT transcription complex subunit, and Sip21, a putative VIP2
protein, are predicted to be components of transcription complex. The CCR4-NOT complex
is an evolutionary conserved protein complex and plays an important role in the control of
transcription and mRNA decay as well as in defense against pathogens (Sarowar et al.,
2007). VIP2 from Avena fatua interacts specifically with A. fatua homologue of maize
transcription factor VIVIPAROUS 1 (AfVP1) which has been implicated in controlling the
maintenance of embryo dormancy in seeds (Jones et al., 2000). The interaction between
Stp1 and Sip16 & Sip21 may suppress plant defense response by influencing transcription
of respective genes. Besides confirming the interaction between Stp1 and its putative
interactors biochemically, the biological significance of these interactions could be
determined by agrobacterium-mediated transient expression of these interactors in N.
benthamiana to test if the expression of these interactors can induce plant defense
responses which, by co-expression with Stp1 are then attenuated again.
Discussion
59
3.3 Stp1, an effector with apoplastic and cytoplasmic functions?
To elucidate the function of secreted effectors, it is mandatory to establish where an
effector localizes after secretion. There are several methods described for localization of
secreted effectors such as heterologous localization in M. oryzae/onion pathosystem, root
uptake assay and biolistic bombardment (Djamei et al., 2011, Kloppholz et al., 2011, Kale
et al., 2010). In addition, there are also in situ translocation assay systems established such
as immunogold electron microscopy, nuclear targeting assay (employing nuclear
localization signal fused proteins) and immunocytochemical technique (Djamei et al.,
2011, Shimada et al., 2009, Khang et al., 2010, Sauer et al., 2006). After transient
expression in maize and N. benthamiana using biolistic bombardment, the C-terminus of
Stp1 as well as full-length Stp1 was localized in the nucleus (Schipper, 2009). Although
advances have been made in the localization assays, the uptake of Stp1 by plant cells has
never been observed to date.
To detect the uptake of Stp1, nuclear targeting assay and immunocytochemical techniques
were used in this study. The fluorescent signal of mCherry could be detected in the nuclei
of SG200∆stp1-stp1-mcherry-HA-NLS infected maize but the signal was very weak and
difficult to quantify because of high background fluorescence (Fig. 25). After
immunocytochemical staining, the signal of Stp1-mCherry-HA-NLS in nuclei was strong
and it could also be observed in the cytoplasm of the plant cell while the blank control
(SG200) and the negative control (SG200pcmu1-mcherry-HA-NLS) produced no specific
signal (Fig. 26). Together with the result that Stp1 was localized in the nucleus when
transiently expressed in plant cells (Schipper, 2009), it revealed that Stp1 may suppress
plant defense responses by affecting transcription of respective genes in the nucleus of
plant cells. The fluorescence signals of Stp1-mCherry-HA-NLS may arise from both Cy3
and mCherry proteins because both the excitation and the emission spectra of Cy3 are close
to mCherry and the mCherry tag may still retain its fluorescent properties after
immunocytochemical staining procedure (Sauer et al., 2006). One advantage of the
immunostain method is that the patchy signal of Cy3 can be easily discriminated from the
evenly distributed autofluorescence. The background problems may be overcome using the
recently established leaf sheath or silk infection (S. Tanaka and R. Kahmann, personal
Discussion
60
communication) (Kankanala et al., 2007). To substantiate the translocation, the localization
of N- and C- terminal domains of Stp1 will also need to be analyzed.
In plants, secreted effectors can either be translocated into the host cell or targeted to the
apoplast (Bozkurt et al., 2012). The preliminary localization assays indicated Stp1 was
translocated in plant cells. Previous study using biolistic bombardment also suggested that
Stp1 might function in plant cells. However, the identification of both apoplastic and
cytoplasmic interactors and the inhibition of Sip3 which is an extracellular effector by the
C-terminus of Stp1 suggested that Stp1 might also function in apoplast. Therefore, Stp1
may distinguish itself among others by functioning as an effector with both apoplastic and
cytoplasmic functions. To substantiate this point the interaction between Stp1 and Sip3
without activation need to be tested. If Stp1 interacts with inactive Sip3, the interaction
could also take place inside the plant cell like the inhibition of cysteine protease, C14 by
AVRblb2 which blocks the secretion of C14 (Bozkurt et al., 2011).
3.4 Glycine-rich domain of Stp1 may promote fungal growth in vascular
bundles.
The deletion of the long variable central domain of Stp1 did not attenuate the colonization
of U. maydis. However this domain exists in all Stp1 orthologs with highly diverse
sequence but similar in length (Fig. 1). Additionally, the middle parts of Stp1 orthologs
from other smut fungi contain repeats which are not present in Stp1 of U. maydis (Fig. 30).
Whether these species specific repeats are functionally relevant or not is not known yet.
Additionally, this long variable domain is a glycine-rich region predicted to be
unstructured. The proteins or protein segments lack secondary and/or tertiary structures are
termed intrinsically disordered (or unstructured) proteins (IDPs/IUPs) (Galea et al., 2008).
It is now widely recognized that IDPs play broad biological roles in all kingdoms of life.
Therefore, the possibility that the central domain of Stp1 might have a minor function in
plant infection could not be rule out.
Although the deletion of the middle part of stp1 did not influence the formation of tumors,
SG200∆stp1 secreting the glycine-rich domain of Stp1 was detected more frequently in
vascular bundles than SG200∆stp1 (Fig. 7). Growing inside vascular bundle is an essential
stage for the infection of Setosphaeria turcica (Chung et al., 2010). The transport of
Discussion
61
conidia of Verticillium spp. in the sap stream of vascular tissues is also essential for the
infection cycle (Fradin & Thomma, 2006). The growth of U. maydis in vascular bundle
may not be important for infection in greenhouse condition where tumors are usually
observed on the leaves but may be important in the field where tumors are mainly formed
on the cob or tassel of maize plants. The colonization of vascular bundle by SG200∆stp1
expressing glycine-rich domain of Stp1 will be substantiated quantitatively by further
confocal microscopy observation.
3.5 The N- and C-terminal domains of Stp1 appear to have distinct functions
In infection with SG200∆stp1 expressing only the N- or C-terminal domains of Stp1, one
would therefore have expected that only a subset of the genes down-regulated in wild type
infection should be down-regulated when infections are carried out with SG200∆stp1-
stp1∆432-515 and SG200∆stp1-stp1∆40-136 and added together this should be similar to the wild
type.
RNA-Seq analysis indicated that 58 up-regulated genes are common to SG200∆stp1,
SG200∆stp1-stp1∆40-136 and SG200∆stp1-stp1∆432-515 (Fig. 28). Besides the genes indicating
metabolic reprogramming, genes involved in plant defense response were identified most
likely representing those triggered by fungal PAMPs (Doehlemann et al., 2008). For
example, 9 of these genes (Table. 11) are identified among the genes involved in early
defense response (Doehlemann et al., 2008). These include among others
GRMZM2G065585, a 1, 3-β-glucanase which can facilitate resistance of rice plants to
fungi infection (Fujikawa et al., 2012), GRMZM2G117989, a Win1 protein which can
Fig. 30. Repeats in the central glycine-rich domain of Stp1 orthologs are absent in U. maydis. S. relianum: Sporisorium reilianum (Schirawski et al., 2010), U. scitamineum: Ustilago scitamineum (R. Kahmann, unpublished), U. hordei: Ustilago hordei (Laurie et al., 2012), U. maydis: Ustilago maydis (Kamper et al., 2006). Blue underline refers to repeats in indicated species. Red dash underline refers to repeat regions in indicated species. Green color denotes conserved amino acids. Pink color denotes variable domain.
Discussion
62
increase the resistance response of Arabidopsis to effector protein, HopW1-1 of P. syringae
(Lee et al., 2008) and GRMZM2G026143, a WIR1A protein, a defense related protein of
wheat (Bull et al., 1992). The induction of these early defense response genes is in line with
the phenotype of SG200∆stp1, SG200∆stp1-stp1∆40-136 and SG200∆stp1-stp1∆432-515 during
infection which stopped after penetration.
3.5.1 Several early defense response genes were not induced by stp1 mutants
expressing the N-terminus of Stp1
The only biological process enriched in plants infected with SG200∆stp1-stp1∆432-515 is
down-regulated cell wall organization or biogenesis process indicating metabolic
reprogramming in infected tissue (Doehlemann et al., 2008). This is common to
SG200∆stp1 and SG200∆stp1-stp1∆432-515 and SG200∆stp1-stp1∆40-136 and could not
disclose specific function of the N-terminus of Stp1. Comparison with early defense genes
(Doehlemann et al., 2008) indicated that 6 early defense genes are identified only in plants
infected with SG200∆stp1 or common to plants infected with SG200∆stp1 and
SG200∆stp1-stp1∆432-515 but are absent in plants infected with SG200∆stp1-stp1∆40-136
(Table. 11). These are for example, GRMZM2G145461, a chitinase which can enhance the
resistance of tobacco plants to biotic and abiotic stress agents (de las Mercedes Dana et al.,
2006), GRMZM2G032910, a epoxide hydrolase 2 which participate in general defense
systems of potato (Mowbray et al., 2006) GRMZM2G092718, a late blight resistance
protein (Doehlemann et al., 2008) and GRMZM2G136372, a thaumatin-like protein which
enhanced the resistance of tobacco plants against fungal pathogen (Munis et al., 2010). The
absence of these early defense response genes suggests that less plant defense response is
triggered by SG200∆stp1 expressing N-terminus of Stp1. However the data is not solid
enough to conclude that N-terminus of Stp1 can partially suppress plant defense responses.
It still needs to be substantiated by Q-PCR to verify the expression of these genes in plants
infected with SG200∆stp1 and SG200∆stp1-stp1∆432-515 and SG200∆stp1-stp1∆40-136.
The putative role of the N-terminus of Stp1in suppressing plant defense response still needs
to be verified. Beside this, the N-terminus of Stp1 might be needed for the uptake of the C-
terminus of Stp1. As exemplified by oomycete secreted effectors Avr1b and AVR3a
(Whisson et al., 2007, Dou et al., 2008) and fungal effectors AvrL567 and AvrM (Kale et
Discussion
63
al., 2010, Rafiqi et al., 2010), the N-terminal domains of secreted effectors are frequently
mediating the translocation of secreted effectors from pathogen to host. The translocation
function of the N-terminus of Stp1 can be verified by localization assays of N- and C-
terminal domains of Stp1. Additionally, as described above, the N-terminus of Stp1 may
function to stabilize the C-terminus of Stp1. This needs to be addressed by future stability
test and IP & Co-IP experiments.
3.5.2 stp1 mutants expressing the C-terminus of Stp1 triggered stronger plant
defense response than stp1 mutants
Besides the maize genes that are common to plants infected with SG200∆stp1,
SG200∆stp1-stp1∆40-136 and SG200∆stp1-stp1∆432-515 indicating metabolic reprogramming
and plant defense response, 95 maize genes are up-regulated only in plants infected with
SG200∆stp1-stp1∆40-136. In these genes, cell wall organization or biogenesis and protein
modification processes are enriched which indicated further metabolic reprogramming.
Interestingly, 7 early defense response genes (Table. 11) identified by microarray
(Doehlemann et al., 2008) are only identified in this subset. For example,
GRMZM2G117971, a Barwin like protein which may involve in a common defense
mechanism in plants (Svensson et al., 1992), GRMZM2G109056, a lipoxygenase which is
a part of the early response of the rice plants to pathogenic attack (Peng et al., 1994),
GRMZM2G416632, a glutathione transferase which is speculated to involved in defense
reactions against pathogens in wheat (Mauch & Dudler, 1993), GRMZM2G106177, an
ergosterol biosynthetic protein which can elicit oxidative burst in tobacco cells
(Kasparovsky et al., 2004) and GRMZM2G044481, a Copalyl Diphosphate Synthase
(AN2) which is induced in maize plants by Fusarium attack (Harris et al., 2005). This
suggests that the scale of plant defense responses induced by SG200∆stp1-stp1∆40-136 is
larger than SG200∆stp1 and SG200∆stp1-stp1∆432-515. Additionally, cell death process is
only enriched in plants infected with SG200∆stp1-stp1∆40-136. Two genes associated with
this cell death process are GRMZM2G028713 and GRMZM2G032602 which both involve
in apoptosis (Supplementary Table. 1). Taken together, stp1 mutant expressing the C-
terminus of Stp1 triggered stronger plant defense response than stp1 mutant.
Discussion
64
The function of the C-terminus is still elusive beside the inhibition of cysteine protease,
Sip3. The preliminary result of RNA-Seq analysis still needs to be verified by Q-PCR and
the stronger plant defense response triggered by the C-terminus of Stp1 needs to be testified
by microscopic observation and overexpression of the C-terminus of Stp1. One possible
mechanism of the stronger plant defense response could be that the C-terminus of Stp1
could partially complement stp1 mutant, thereafter, more biomass was produced and
stronger plant defense response was induced. Another possibility is that the N-terminus of
Stp1 might modify the C-terminus of Stp1 and when this modification is absent stronger
plant defense response could be induced.
3.6 Working model of the function of Stp1
A working model is proposed based on current results (Fig. 31). After formation of the
dikaryotic hyphae, Stp1 starts to be expressed and secreted in a glycosylated form to the
apoplast of maize cells. In the apoplast, Stp1 can inhibit a class of papain-like cysteine
proteases. Meanwhile, Stp1 may also be cleaved into separate N- and C- terminal domains
in the apoplast. The inhibition of cysteine proteases may not be the only function of Stp1.
Stp1 may be taken up by maize cells. In the plant cytosol, Stp1 may bind to and block the
secretion of maize papain-like cysteine proteases. Moreover, Stp1 may also translocate to
the nucleus of maize cells to suppress plant defense responses by affecting transcription of
respective genes.
Several key challenges still need to be addressed to substantiate this preliminary model.
First of all, the biological significance of the inhibition of cysteine proteases by Stp1 needs
to be determined. Secondly, the interaction between Stp1 and the maize cytoplasmic
interaction partners needs to be confirmed. Finally, the location of the full length Stp1 as
well as N- and C-terminal domains during infection needs to be determined.
Discussion
65
Fig. 31. Working model of the function of Stp1 : Stp1, : Sip3 and other maize secreted cysteine proteases.
Materials and methods
66
4. Materials and methods
4.1 Materials and source of supplies
4.1.1 Chemicals and enzymes
All chemicals used in this study were obtained from Difco (Augsburg), Fisher Scientific
(Schwerte), Fluka (Buchs), GE Healthcare (München), GERBU Biochemicals (Gaiberg),
IBA (Göttingen), Life Technologies (Darmstadt), Merck (Darmstadt), Millipore
(Schwalbach/Ts), NEB (Frankfurt am Main), PeptaNova (Sandhausen), QIAGEN
(Hilden), Roche (Mannheim), Roth (Karlsruhe), Clontech (Saint-Germain-en-Laye) and
Sigma-Aldrich (Deisenhofen).
All restriction enzymes, Taq DNA polymerase, T4 DNA ligase, T4 PNK was obtained from
NEB (Frankfurt am Main). Phusion high-fidelity DNA polymerase was obtained from
Finnzymes (Frankfurt am Main). KOD extreme polymerase was obtained from Merck
(Darmstadt), Gateway clonase was obtained from Invitrogen (Karlsruhe), Prescission
protease was obtained from GE Healthcare (München), TEV protease was obtained from
Promega (Mannheim).
4.1.2 Buffers and solutions
Standard buffers and solutions are prepared according to Ausubel et al. (1987) and
Sambrook et al (1989). Specific buffers and solutions are listed with the corresponding
methods. All media, solutions and buffers were autoclaved for 5 min at 121 °C. Heat-
sensitive solutions were filter-sterilized (pore size: 0.2 μm, Merck, Darmstadt).
4.1.3 Kits
The following kits were used following protocols recommended by the suppliers:
TOPO TA cloning kit (Invitrogen, Karlsruhe) for direct cloning of PCR products, Wizard
SV gel and PCR clean-up system (Promega, Mannheim) for extraction and purification of
DNA fragments, QIAquick plasmid purification kit (QIAGEN, Hilden) for plasmid
isolation and purification, ECL Plus Western Blot detection reagent (GE Healthcare,
München) was used for chemiluminescence detection. QuikChange Multi Site-Directed
mutagenesis kit (Agilent Technologies, Böblingen) for site-directed mutagenesis of plasmid
Materials and methods
67
DNA. ProLong® antifade kit (Life Technologies, Darmstadt) for protection of the dyes
from fading during fluorescence microscopy.
4.2 Media
4.2.1 Media for E. coli and A. tumefaciens
E. coli and A. tumefaciens strains were grown in dYT liquid medium or YT solid medium
supplemented with appropriate antibiotics (Ampicillin (Amp), 100 μg/ml; Kanamycin
(Kan), 50 μg/ml; Rifampicin (Rif), 50 μg/ml). dYT glycerol medium was use for
preparation of frozen stocks.
dYT liquid medium (Sambrook et al., 1989) 1.6 % (w/v) Trypton-Pepton
1.0 % (w/v) Yeast Extract
0.5 % (w/v) NaCl
Add dH2O and autoclave
YT solid medium 0.8 % (w/v) Trypton-Pepton
0.5 % (w/v) Yeast Extract
0.5 % (w/v) NaCl
1.3 % (w/v) Agar
Add dH2O and autoclave
dYT glycerol medium 1.6 % (w/v) Trypton-Pepton
1.0 % (w/v) Yeast Extract
0.5 % (w/v) NaCl
800 ml (v/v) 87 % Glycerol (f.c.
69.6 %)
Add dH2O and autoclave
4.2.2 Media for U. maydis
U. maydis strains were grown on potato-dextrose-agar (Difco), YEPSlight liquid media or
regeneration agar supplemented with carboxin (2 μg/ml). NSY-glycerol was used for
preparation of frozen stocks.
Materials and methods
68
Potato-Dextrose-Agar (PD) 2.4 % (w/v) Potato-Dextrose Broth
2 % (w/v) Bactoagar
Add dH2O and autoclave
YEPSlight (modified from Tsukuda et al., 1988) 1 % (w/v) Yeast-Extract
1 % (w/v) Pepton
1 % (w/v) Saccharose
Add dH2O and autoclave
NSY-glycerol 0.8 % (w/v) Nutrient Broth
0.1 % (w/v) Yeast-Extract
0.5 % (w/v) Saccharose
69.6 % (v/v) Glycerol
Add dH2O and autoclave
Regeneration Agar (Schulz et al., 1990) 1.5 % (w/v) Agar
1 M Sorbitol
in YEPSL (described above)
Add dH2O and autoclave
4.2.3 Media for S. cerevisiae
Yeast strains were grown on YEPD-medium or SD-medium supplemented with appropriate
nutrient (Table. 12).
YEPD-medium 2 % (w/v) Pepton (Difco)
1 % (w/v) Yeast extract
2 % (w/v) Agar
2 % (w/v) Glucose (after autoclave)
0,003 % (w/v) Adenine (after
autoclave)
SD-medium 0,67 % (w/v) Yeast nitrogen base
without amino acids
0.06 % (w/v) –Ade/-His/-Leu/-Trp
Materials and methods
69
Do Supplement
2 % (w/v) Agar (for plate only)
2 % (w/v) Glucose (after autoclave)
Nutrients 10×Concentration
L-Adenine hemi-sulfate salt 200 mg/L
L-Histidine HCl monohydrate 200 mg/L
L-Leucine 1000 mg/L
L-Tryptophan 200 mg/L
4.3 Strains
4.3.1 Escherichia coli strains
E. coli strains K-12 TOP10 ([F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacO74 recA1araΔ139 Δ(ara-leu)7697 galU
galKrpsL (StrR) endA1 nupG], Invitrogen, Karlsruhe) and K-12 DH5α ([F- Φ80d lacZ ΔM15 Δ(lacZYA-argF) U169 deoR
recA1 endA1 hsdR17(rK-, mK+) phoA supE44 λ- thi-l gyrA96 relA1], Gibco/BRL, Eggenstein) were used as host strains
for plasmid constructions and amplifications. E. coli strains BL21 Rosetta (DE3)pLysS( [F-
ompT hsdSB(rB-mB-) gal dcm (DE3) pLacIRARE (CamR)], Novagen/Merck, Darmstadt) was used as host strains to
produce recombinant proteins.
4.3.2 Agrobacterium tumefaciens strain
A. tumefaciens GV3101 stain is used for transient expression of protein in Nicotiana
benthamina. A. tumefaciens GV3101 expressing p19 protein, a suppressor of gene silencing
is used for co-infiltration of N. benthamiana with A. tumefaciens GV3101 expressing target
proteins (Voinnet et al., 2003).
4.3.3 Ustilago maydis strains
All U. maydis strains used in this study are listed in table. 13
Strain Genotype Antibiotics Reference
SG200 a1mfa2bE1bW2 Phleo (Kamper et al., 2006)
SG200∆stp1 a1mfa2bE1bW2 Δum02475::egfp Phleo, Hyg (Schipper, 2009)
SG200∆stp1-stp1 a1 mfa2 bE1 bW2 Δum02475 Phleo, Hyg, (Schipper, 2009)
Table 13. U. maydis strains used in this study
Table 12. Nutrients supplemented in SD-medium
Materials and methods
70
ipr [Pstp1:stp1] ips Cbx
SG200∆stp1-stp1Δ136-432 a1 mfa2 bE1 bW2 Δum02475
ipr [Pstp1: stp1Δ136-432] ips
Phleo, Hyg,
Cbx
This study
SG200∆stp1-stp1∆29-
136/432-515
a1 mfa2 bE1 bW2 Δum02475
ipr [Pstp1: stp1∆29-136/432-515] ips
Phleo, Hyg,
Cbx
This study
SG200∆stp1-stp1∆432-515 a1 mfa2 bE1 bW2 Δum02475
ipr [Pstp1: stp1∆432-515] ips
Phleo, Hyg,
Cbx
(Schipper, 2009)
SG200∆stp1-stp1∆40-136 a1 mfa2 bE1 bW2 Δum02475
ipr [Pstp1: stp1∆40-136] ips
Phleo, Hyg,
Cbx
(Schipper, 2009)
SG200∆stp1-stp1∆137-
515+stp1∆37-431
a1 mfa2 bE1 bW2 Δum02475
ipr [Pstp1: stp1∆137-515+ Pstp1:
stp1∆37-431] ips
Phleo, Hyg,
Cbx
This study
SG200∆stp1-stp1Δ136-
432T452V
a1 mfa2 bE1 bW2 Δum02475
ipr [Pstp1: stp1Δ136-432T452V] ips
Phleo, Hyg,
Cbx
This study
SG200∆stp1-stp1T452V a1 mfa2 bE1 bW2 Δum02475
ipr [Pstp1: stp1T452V] ips
Phleo, Hyg,
Cbx
This study
SG200∆stp1-stp1PPAA a1 mfa2 bE1 bW2 Δum02475
ipr [Pstp1: stp1PPAA] ips
Phleo, Hyg,
Cbx
This study
SG200∆stp1-stp1SRAA a1 mfa2 bE1 bW2 Δum02475
ipr [Pstp1: stp1SRAA] ips
Phleo, Hyg,
Cbx
This study
SG200∆stp1-stp1Δ433-454 a1 mfa2 bE1 bW2 Δum02475
ipr [Pstp1: stp1Δ433-454] ips
Phleo, Hyg,
Cbx
This study
SG200∆stp1-stp1Δ455-476 a1 mfa2 bE1 bW2 Δum02475
ipr [Pstp1: stp1Δ455-476] ips
Phleo, Hyg,
Cbx
This study
SG200∆stp1-stp1Δ477-494 a1 mfa2 bE1 bW2 Δum02475
ipr [Pstp1: stp1Δ477-494] ips
Phleo, Hyg,
Cbx
This study
SG200∆stp1-stp1Δ495-515 a1 mfa2 bE1 bW2 Δum02475
ipr [Pstp1: stp1Δ495-515] ips
Phleo, Hyg,
Cbx
This study
SG200∆stp1-Uh-stp1 a1 mfa2 bE1 bW2 Δum02475
ipr [Pstp1: Uh-stp1] ips
Phleo, Hyg,
Cbx
This study
SG200∆stp1-Us-stp1 a1 mfa2 bE1 bW2 Δum02475
ipr [Pstp1: Us-stp1] ips
Phleo, Hyg,
Cbx
This study
SG200∆stp1-Stp1-
cherry-HA-NLS
a1 mfa2 bE1 bW2 Δum02475
ipr [Pcmu: stp1-cherry-HA-NLS]
ips
Phleo, Hyg,
Cbx
This study
SG200Um05731- a1 mfa2 bE1 bW2 Δum02475 Phleo, Hyg, Armin Djamei (This lab)
Materials and methods
71
cherry-HA-NLS ipr [Pcmu: um05731-cherry-HA-
NLS] ips
Cbx
SG200-cherry-HA-NLS a1 mfa2 bE1 bW2 Δum02475
ipr [Pcmu: cherry-HA-NLS] ips
Phleo, Hyg,
Cbx
This study
SG200∆stp1-stp1∆136-
432-HA-NLS
a1 mfa2 bE1 bW2 Δum02475
ipr [Pcmu: stp1∆136-432-HA-NLS]
ips
Phleo, Hyg,
Cbx
This study
SG200-HA-NLS a1 mfa2 bE1 bW2 Δum02475
ipr [Pcmu: HA-NLS] ips
Phleo, Hyg,
Cbx
This study
4.3.4 Saccharomyces cerevisiae strains
S. cerevisiae strain AH109 (MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4Δ, gal80Δ,
LYS2::GAL1UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2, RA3::MEL1UASMEL1TATA-lacZ,
MEL1, Clontech) was used as host strain for yeast two-hybrid assay.
4.4 Oligonucleotides
All oligonucleotides used in this study were ordered from Eurofins MWG Operon (Table.
14).
primer Cleavage
site*
Sequence
LL7 AACATGACCATGGCAGCCGCCCCTTCTCGTTAGGCG
LL8 ATGGCACCTCCTCCTGCCGCCTAGGCGCGGCCGCCC
LL15 ATGCGAGCCGACTTTTTATCC
LL21 ATGAGAGCCGTGCTCTCGCTCAAC
LL23 NcoI CATGCCATGGCTGCACTGCAACCC
LL24 NotI ATAAGAATGCGGCCGCTTTTCTGAGCTGCATAGCTTTCTG
LL25 SfiI CTCGGCCGGTGCGGCCGTGCTGATGTGCATCAATGGCGATCCTGCTG
LL30 NcoI CATGCCATGGCAAGGTCAGGCTTAG
LL31 NotI ATAAGAATGCGGCCGCTTCTAAGGTTTGGTGATGTTGAAG
LL32 NcoI CATGCCATGGAAAGGTACAAATTGATACG
LL33 NotI ATAAGAATGCGGCCGCTGAAGAAATTCACCTGCCG
LL56 ATGAGGGCCAAACTTTCGCTCAAC
LL61 BspHI CAAGTCATGATAATGGGCCCTCCGCGGCCGG
Table 14. Oligonucleotides used in this study
Materials and methods
72
LL62 NotI ATAAGAATGCGGCCGCTGCAGCCGCACGGTCCCTCC
LL66 NotI ATAAGAATGCGGCCGCTTGCGCTGCTCTTCATGCCATC
LL67 BspHI CAAGTCATGATAGCGGACAGCATGTCGATCGTCTC
LL68 CAAGTTCAACACCATGACTGGTGTTCGCATCCGC
LL72 GGGATCCATCGAGCTCGAG
LL73 GGGAACATCAGCTGGGGG
LL74 GTGCTGATGTGCATCAATGG
LL75 CATATGAGCGTAATCTGGTACGTC
LL76 GGGATCCGTCGACCTGC
LL77 CATATGCAGGTCCTCCTCTG
LL78 GGCCTGAACGGCGACAAGTATCGGCAGG
LL79 TAGGCGCGGCCGCCCGGC
LL80 AAGCAAGCTGATGGAGGAGCAGGTACG
LL81 TTTCGGAGCATTTGCGTTTGAAGCGGG
LL87 GGCGCCGGCGCCCAGGAGGAT
LL88 NotI ATAAGAATGCGGCCGCTCATCCGAGGGATGTGCAGTGGACGGAAGG
LL89 GGACAACATGTGTTTACGGAGC
LL90 NotI ATAAGAATGCGGCCGCTCATCCGTGGCGAGTGCATTT
LL93 GGCGCCGGCGCCAACAAC
LL94 NotI ATAAGAATGCGGCCGCTCATGCCACGCCCCATGGTCTGCTC
LL97 GTTCTGATGTGTATTAATGGTGATCCGGC
LL98 CGGAACATCTGCCGGAGGTGTAAC
LL112 CGTGTTCTCTGCTTTATTTTCTCGATAAAGTTGTGG
LL113 NotI GCAGCCGGGCGGCCGCGCTTATTGCTTCGGAGGGGGG
LL114 NotI GCAGCCGGGCGGCCGCGCCTAACGAGCAGGAGGAGGG
LL116 CGTAACCTAGAGCTCTTGCAGTTCG
LL117 AAGATCCGCTCGATCGCCACC
LL119 ACGAGAAGGAGGAGGTGCCATGGT
LL157 TAAAAGCTTGATCCGGCTGCTAACAAAG
LL158 GGATCCCTGAAAATACAGGTTTTCGGTC
LL159 BamHI CGGGATCCATGCACACGCTCGATATCCATTC
LL160 HindIII TCCCAAGCTTCTAGCTTACAAACTTGTTGCTCTGC
LL175 GGCTGGGCCCATGGGCTGGAG
LL176 Not I ATAAGAATGCGGCCGCTCAGAGGGTACGACGGCTCAACGGCGATAC
LL177 CACCCGCAGTTCGAAAAAGCGGACAGCATGTCGATCGTCTC
LL178 PspOMI GCTCCAGCCCATGGGCCCAGCCTGCTTTTTTGTACAAAGTTGGC
LL179 TTCCAGATTACGCTGGCGGCATGGACGAGCTGTACAAGTACCC
Materials and methods
73
LL180 CATCGTATGGGTAGGTGGCGATCGAGCGGATCTTACGAGAAGG
LL181 CATCGTATGGGTAGGTGGCGATCGAGCGGATCTTGGCCTGAAC
oKS145 SfiI GAGGGCCGCACCGGCCCCGGGAACATCAGCTGGGGGAGTAACGTGGATGC
*, The cleavage sites of primers were underlined
4.5 Plasmids
4.5.1 Plasmids for generation of U. maydis mutants
p123_L-egfp, p123 (Aichinger et al., 2003) derivative containing the carboxin resistance gene and an eGFP gene which is fused to the otef promoter and nos terminator.
p123-Pstp1-stp1, p123_L-egfp derivative incorporating stp1 and its native promoter(Schipper, 2009).
p123pstp1-stp1Δ136-432, This plasmid was generated through inverse PCR using LL25 and oKS 145 as primers and p123-Pstp1-stp1 as template. The PCR product was digested by SfiI and ligated to generate p123pstp1-stp1.
p123pstp1-Stp1∆29-136/432-515, The vector of this plasmids were obtained through inverse PCR employing LL78 and LL 79 as primers and p123-Pstp1-stp1 as template. The stp1∆29-136/432-515 fragment was amplified using LL80 and LL81 as primers and p123-Pstp1-stp1 as template. The fragments were treated with T4 PNK (NEB) and ligated to produce p123pstp1-Stp1∆29-136/432-515.
p123pstp1-stp1∆137-515+pstp1-stp1∆37-431, This plasmid was constructed by Michael Daume and Kerstin Schipper. It incorporates two gene fragments, encoding N- or C-termini of Stp1, which fused with promoter of stp1 separately.
p123pstp1-stp1Δ136-432T452V, The point mutation was introduced through inverse PCR using QuikChange Multi Site-Directed Mutagenesis Kit using LL68 as primers and p123pstp1-stp1Δ136-432 as a template. The PCR product was treated with T4 PNK and self-ligated to produce p123pstp1-stp1Δ136-432T452V.
p123pstp1-stp1T452V, The point mutation was introduced through inverse PCR using QuikChange Multi Site-Directed Mutagenesis Kit using LL68 as primers and p123-Pstp1-stp1 as template. The PCR product was treated with T4 PNK and self-ligated to produce p123pstp1- stp1Δ136-432T452V.
p123pstp1-stp1PPAA, The point mutation was introduced through inverse PCR using QuikChange Multi Site-Directed Mutagenesis Kit using LL7 as primers and p123-Pstp1-stp1 as template. The PCR product was treated with T4 PNK and self-ligated to produce p123pstp1-stp1PPAA.
p123pstp1-stp1SRAA, The point mutation was introduced through inverse PCR using QuikChange Multi Site-Directed Mutagenesis Kit using LL8 as primers and p123-Pstp1-stp1 as template. The PCR product was treated with T4 PNK and self-ligated to produce p123pstp1-stp1SRAA.
p123pstp1-stp1Δ433-454, This plasmid was constructed by Kerstin Schipper. It incorporates C-terminus truncated Stp1.
p123pstp1-stp1Δ455-476, This plasmid was constructed by Kerstin Schipper. It incorporates C-terminus truncated Stp1.
p123pstp1-stp1Δ477-494, This plasmid was constructed by Kerstin Schipper. It incorporates C-terminus truncated Stp1.
p123pstp1-stp1Δ495-515, This plasmid was constructed by Kerstin Schipper. It incorporates C-terminus truncated Stp1.
p123pstp1-Uh-stp1, The vector was amplified using LL25 and LL112 as primers and p123-Pstp1-stp1 as template. The Uh-stp1(stp1 from U. hordei) was amplified using LL15 and LL113 as primers and genome DNA of U. hordei as template. Both
Materials and methods
74
the fragment and the vector were treated with T4 PNK before digestion by NotI. Ligate the vector and the fragment to produce p123pstp1-Uh-stp1.
p123pstp1-Us-stp1, The vector was amplified using LL25 and LL112 as primers and p123-Pstp1-stp1 as template. The Us-stp1 (stp1 from U. scitaminum) was amplified using LL56 and LL114 as primers and genome DNA of U. scitaminum as template. Both the fragment and the vector were treated with T4 PNK before digestion by NotI. The vector and the fragment was ligated to produce p123pstp1-Us-stp1.
p123pcmu-stp1-cherry-HA-NLS, Stp1 fragment was amplified using LL 21 and LL119 as primers and p123-Pstp1-stp1 as template. The vector containing mCherry, HA and NLS was amplified through inverse PCR using LL116 and LL117 as primers and p123-Um05731genomic-cherry (From Armin Djamei). The fragment and the vector were treated with T4 PNK and ligated to produce p123pcmu-Stp1-Cherry-HA-NLS.
P123pcmu-cherry-HA-NLS, The plasmid was constructed through inverse PCR using p123pcmu-Stp1-Cherry-HA-NLS as template and LL78 & LL117 as primers. The PCR product was treated with T4 PNK and self-ligated to produce P123pcmu-Cherry-HA-NLS.
P123pcmu-stp1∆136-432-HA-NLS, The vector was amplified using LL179 and oKS145 as primers and p123pcmu-Stp1-Cherry-HA-NLS as template. The stp1 fragment was amplified using LL25 and LL180 as primers and p123pcmuStp1-Cherry-HA-NLS as template. Both the vector and the stp1 fragment were digested by SfiI and treated with T4 PNK. The vector and the stp1 fragment were then ligated tp generate P123pcmu-stp1∆136-432-HA-NLS.
P123pcmu-HA-NLS, The plasmid was constructed through inverse PCR using LL179 and LL181 as primers and P123pcmu-Cherry-HA-NLS as template. The PCR product was treated with T4 PNK and self-ligated to produce P123pcmu-HA-NLS.
4.5.2 Plasmids for Y2H assays
pGADT7, AD/library cloning vector incorporating HA epitope and LEU2 selection marker (Clontech).
pGBKT7, DNA/bait cloning vector incorporating c-Myc epitope and TRP1 selection marker (Clontech).
pGAD-stp1, stp1 inserted into pGADT7 vector (Schipper, 2009).
pGBK-stp1, stp1 inserted into pGBKT7 vector(Schipper, 2009).
pGBK-stp1∆136-338, stp1∆136-338 inserted into pGBKT7 vector (Schipper, 2009).
pGAD-sip10, sip10 fragment inserted into pGAD vector(Schipper, 2009).
pAD, AD/library cloning vector incorporating HA epitope and LEU2 selection marker (Stratagene, LaJolla/USA).
pAD-sip12, sip12 fragment inserted into pAD vector (Clontech).
pGAD-sip29, sip29 fragment inserted into pGAD vector (Schipper, 2009).
pGAD-sip31, sip31 fragment inserted into pGAD vector(Schipper, 2009).
pGBK-stp1∆136-432, This plasmid was generated through inverse PCR using LL25 and oKS 145 as primers and pGBK-stp1 as template. The PCR product was digested by SfiI and ligated to generate pGBK-stp1∆136-432.
pGADGW, gateway destination vector modified from pGADT7 by Armin Djamei (this lab).
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75
pGBKGW, gateway destination vector modified from pGBKT7 by Armin Djamei (this lab).
pEntry 4B Vcp1, gateway entry vector incorporating BspHI/NcoI and NotI cleavage sites modified from pENTR TOPO vector (invitrogen) by Armin Djamei (this lab).
pGAD-sip6, sip6 fragment was amplified using LL61 and LL62 as primers and cDNA of maize as template. Both sip6 fragment and pEntry 4B Vcp1 vector were digested by BspHI and NotI. The fragments was purified and ligated to generate pEntry-sip6. Sip6 fragment was transferted into pGADGW vector through an LR recombination reaction.
pGAD-sip8, sip8 fragment was amplified using LL23 and LL24 as primers and cDNA of maize as template. Both sip8 fragment and pEntry 4B Vcp1 vector were digested by NcoI and NotI. The fragments was purified and ligated to generate pEntry-sip8. sip8 fragment was transferted into pGADGW vector through an LR recombination reaction.
pGAD-sip9, sip9 fragment was amplified using LL93 and LL94 as primers and cDNA of maize as template. pEntry 4B Vcp1 vector was digested by NcoI and the gap was filled by DNA polymerase I large (Klenow) fragment. Both sip9 fragment and pEntry 4B Vcp1 fragment were digested by NotI. The fragments were purified and ligated to generate pEntry-sip9. Sip9 fragment was transferted into pGADGW vector through an LR recombination reaction.
pGAD-sip14, sip14 fragment was amplified using LL87 and LL88 as primers and cDNA of maize as template. pEntry 4B Vcp1 vector was digested by NcoI and the gap was filled by DNA polymerase I large (Klenow) fragment. Both sip14 fragment and pEntry 4B Vcp1 fragment were digested by NotI. The fragments were purified and ligated to generate pEntry-sip14. Sip14 fragment was transferted into pGADGW vector through an LR recombination reaction.
pGAD-sip16, sip16 fragment was amplified using LL30 and LL31 as primers and cDNA of maize as template. Both sip16 fragment and pEntry 4B Vcp1 vector were digested by NcoI and NotI. The fragments was purified and ligated to generate pEntry-sip16. Sip16 fragment was transferted into pGADGW vector through an LR recombination reaction.
pGAD-sip19, sip19 fragment was amplified using LL32 and LL33 as primers and cDNA of maize as template. Both sip19 fragment and pEntry 4B Vcp1 vector were digested by NcoI and NotI. The fragments was purified and ligated to generate pEntry-sip19. Sip19 fragment was transferted into pGADGW vector through an LR recombination reaction.
pGAD-sip21, sip21 fragment was amplified using LL89 and LL90 as primers and cDNA of maize as template. pEntry 4B Vcp1 vector was digested by NcoI and the gap was filled by DNA polymerase I large (Klenow) fragment. Both sip21 fragment and pEntry 4B Vcp1 fragment were digested by NotI. The fragments were purified and ligated to generate pEntry-sip21. Sip21 fragment was transferted into pGADGW vector through an LR recombination reaction.
pGAD-sip3, sip3 fragment was amplified using LL66 and LL67 as primers and cDNA of maize as template. Both sip3 fragment and pEntry 4B Vcp1 vector were digested by BspHI and NotI. The fragments was purified and ligated to generate pEntry-sip3. Sip3 fragment was transferted into pGADGW vector through an LR recombination reaction.
pGAD-stp1∆136-432, This plasmid was generated through inverse PCR using LL25 and oKS 145 as primers and pGAD-stp1 as template. The PCR product was digested by SfiI and ligated to generate pGAD-stp1∆136-432.
pGBK-stp129-135, This plasmid was generated through inverse PCR using LL76 and LL77 as primers and pGBK-stp1 as template. The PCR product was treated with T4 PNK (NEB) and ligated to produce pGBK-stp129-135.
pGAD-stp129-135, This plasmid was generated through inverse PCR using LL72 and LL73 as primers and pGAD-stp1 as template. The PCR product was treated with T4 PNK (NEB) and ligated to produce pGAD-stp129-135.
pGBK-stp1433-515, stp1433-515 inserted into pGBKT7 vector (Schipper, 2009).
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pGAD-stp1433-515, This plasmid was generated through inverse PCR using LL74 and LL75 as primers and pGAD-stp1 as template. The PCR product was treated with T4 PNK (NEB) and ligated to produce pGAD-stp1433-515.
pGAD-Mir3, Mir3 inserted into pGADT7 vector constructed by Nina Neidig (this lab).
pGAD-CP1A, CP1A inserted into pGADT7 vector (A. Müller this department, personal communication).
pGAD-CP2, CP2 inserted into pGADT7 vector (A. Müller, personal communication).
pGAD-XCP2, XCP2 inserted into pGADT7 vector (A. Müller, personal communication).
pGAD-CatB3, CatB3 inserted into pGADT7 vector (A. Müller, personal communication).
4.5.3 Plasmids for protein expression
pRSET His TEV, Expression vector for E. coli incorporating His-tag and TEV protease cleavage site(Schoepfer, 1993).
pBIN19AN-3CI-YFP, a plant binary vector derived from pBI121(Haseloff et al., 1997, Jefferson et al., 1987). Expression vector for agrobacterium mediated expression in tobacco, incorporating IgG-tag and prescission protease cleavage site(From Nina Neidig, this lab).
pMA Stp1∆136-432, Codon optimized Stp1∆136-432 for E. coli inserted in pMA vector(GENEART).
pBIN Strep sip3WG, pEntry strep sip3 was constructed through inverse PCR employing LL177 and LL178 as primers and pEntry-sip3 as template. The fragment was treated with T4 PNK (NEB) and ligated to produce pEntry strep sip3. Sip3WG fragment was amplified through PCR using LL175 and LL 176 as primers and pEntry strep sip3 as template. sip 3WG fragment and pBIN PP vector were digested by Bsp120I & NotI and ligated to produce pBIN strep sip3WG
pRSET His TEV Stp1∆136-432, Stp1∆136-432 fragment was obtained from pMA Stp1∆136-432 by digestion using BamHI and HindIII. The vector was generated by digestion using BamHI and HindIII. The fragment and the vector were ligated to produce pRSET His TEV Stp1∆136-432.
pRSET His TEV Stp129-135, This plasmid was generated through inverse PCR using pRSET His TEV Stp1∆136-432 as template and LL98 and LL157 as primers. The PCR product was treated with T4 PNK (NEB) and ligated to produce pRSET His TEV Stp129-135.
pRSET His TEV Stp1433-515, This plasmid was generated through inverse PCR using pRSET His TEV Stp1∆136-432 as template and LL97 and LL158 as primers. The PCR product was treated with T4 PNK (NEB) and ligated to produce pRSET His TEV Stp1433-515.
pRSET His TEV Aro7, Aro7 fragment was amplified from genomic DNA of SG200 using LL159 and LL160 as primers. Both the PCR product and pRSET His TEV were digested by BamHI and HindIII. The purified and fragment and vector were ligated to generate pRSET His TEV Aro7.
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4.6 Microbiological methods
4.6.1 E. coli and A. tumefaciens methods
Liquid cultures were incubated at 37 °C (E. coli) or 28 °C (A. tumefaciens) at 200 rpm.
Solid media were incubated under aerobic condition at 37 °C (E. coli) or 28 °C (A.
tumefaciens). To prepare frozen stocks, exponentially growing cultures were mixed with
dYT glycerol medium at a 1:1 ratio and stored at -80 °C.
4.6.1.1 Preparation of chemical competent cells and transformation of E. coli
The chemical competent cells were prepared following the protocol of Hanahan (Hanahan,
1985). To transform the E. coli, an aliquot of competent cells was thawed on ice.
Afterwards, 1-10 μl plasmid or ligation mixture was added, gently mixed and incubated on
ice for 15-30 min. The mixture was then heat shocked at 42 °C for 1 min and immediately
cooled on ice for 30 sec. For the recovery of the E. coli cells, 300 µl dYT medium was
added and the cells were incubated at 600 rpm for 30-60 min at 37 °C. Finally, the entire
E. coli cell suspension was plated on YT-agar containing appropriate antibiotics and
incubated at 37 °C overnight.
4.6.1.2 Preparation of electro-competent cells and transformation of A. tumefaciens
Transformation by electroporation was used for E. coli as well as for A. tumefaciens. A
fresh over night culture was diluted at a ratio 1:100 in 400ml dYT liquid medium and
shaked at 200 rpm until OD600 ≈ 0.7. The cells were then cooled on ice for 15-30 min and
centrifuged at 3000 rpm 15 min 4 °C. Afterwards, the pellet was washed by 200 ml distilled
H2O twice. Subsequently the pellet was washed by 10 ml 10 % glycerol and resuspended in
0.5-1.0 ml 10% glycerol. Finally, 50 μl of the competent cells were aliquoted into pre-
chilled 1.5 ml microcentrifuge tubes for immediate use, or stored at -80 °C for later use.
For transformation, the electro-competent cells were thawed on ice. Up to 5 µl DNA was
added, gently mixed and transferred into a pre-chilled 0.2 cm electroporation cuvette. The
mixture was then placed into the holder on the gene pulser (E. coli pulser Bio-Rad) and the
pulse was directly initiated (25 mF, 200 W, 2.0 kV for E. coli and 1.6 kV for A.
tumefaciens). After discharge (hold the button for 4-5 sec), 1ml dYT liquid medium was
pipetted into the transformation mixture. Subsequently, the mixture was transferred into a
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78
1.5 ml microcentrifuge tube and the cells were incubated at 600 rpm for 30-60 min.
Finally, the entire cell suspension was plated on YT-agar containing appropriate antibiotics.
4.6.1.3 TOPO TA cloning
DNA fragments were amplified through PCR using Taq polymerase. The PCR product was
then ligated with the TOPO TA cloning vector following the standard protocol (Invitrogen)
and transformed into E. coli cells as described above. The transformant with blue colony
were selected. The plasmids were extracted using the QIAquick plasmid purification kit
following the standard protocol and finally verified by restriction enzyme digestion.
4.6.1.4 Expression of His-Stp1∆136-432, His-Stp1433-515 and His-Aro7 in E. coli
A pre-culture of E. coli BL21 cells containing plasmids encoding corresponding proteins
were grown overnight. The culture was diluted at a ratio 1:100 in 400 ml dYT liquid
medium containing appropriate antibiotics and grown until OD600≈1.0. The protein
expression was induced by 0.5 mM IPTG. The temperature was then shifted to 20 °C and
the induction was continued for ~ 20 hours.
4.6.1.5 N. benthamiana infiltration for protein expression
The pre-culture of A. tumefaciens strains containing p19 and the plasmids encoding
corresponding proteins were grown overnight. The culture was diluted at a ratio 1: 20 in 50
ml dYT liquid medium containing appropriate antibiotics and grown until OD600≈1.0
(about 4 hours). The cells were harvested by centrifuging at 6000 rpm, 5min, and room
temperature. Then, the cells were washed three times by MES buffer. Afterwards, the
pellets were re-suspended in MES buffer supplemented with 150 µM acetosyringone and
the cell density was adjusted to OD600≈1.0. The cells suspension of A. tumefaciens strains
containing p19 and the plasmids encoding corresponding proteins were mixed for
infiltration of N. benthamiana.
For infiltration, 2-3 weeks (after singling out of the seedlings) old N. benthamiana was
used. The A. tumefaciens mixture was infiltrated into the back side of the young leaves
using 1ml syringe without the needle. For each plant, two young leaves were infiltrated.
The infiltrated leaves were harvested by immediate frozen in liquid nitrogen and stocked in
-80 °C for protein purification.
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79
MES 10 mM MES, pH 5.6
10 mM MgCl2
Add dH2O and autoclave
4.6.2 U. maydis methods
Liquid cultures were incubated at 28 °C at 200 rpm. Solid media were incubated under
aerobic condition at 28 °C. The cell density of culture was determined using a Novosec II
Photometer (Pharmacia Biotech) at an optical density of 600 nm (OD600). The
corresponding culture medium was used as a reference. A culture density of OD600 ≈1.0
corresponds to about 1-5 ×107 cells/ml. To prepare frozen stocks, exponentially growing
cultures were mixed with NSY-glycerol at a 1:1 ratio and stored at -80 °C.
4.6.2.1 Protoplast preparation and transformation of U. maydis
Protoplast preparation and transformation of U. maydis was performed as described by
Schulz (Schulz et al., 1990). Pre-culture of U. maydis cells were grown in YEPSlight
medium for at least 8 hours. The culture was diluted in 50 ml YEPSlight medium at a ratio
1:300 to 500 and grown over night to OD600 0.5-0.8. Cells were harvested by
centrifugation at room temperature for 5 min at 3500 rpm, washed in 25 ml SCS, and re-
suspended in 2 ml SCS containing 2.5 mg/ml Novozyme. Cells were incubated for 5-10
min at room temperature until about 50 % of the cells are beginning to protoplast, which
was monitored under the microscope. Afterwards, U. maydis cells were washed three times
with 20 ml SCS and centrifuged at 2300 rpm for 5 min at room temperature. This was
followed by an additional wash with 20 ml STC and centrifuged at 2400 rpm for 5 min at
room temperature. Finally, protoplast pellets were re-suspended in 0.5 ml of ice cold STC,
and 70 μl of protoplasts were aliquoted into pre-chilled 1.5 ml microcentrifuge tubes for
immediate use, or stored at -80 °C for later use.
For transformation of protoplasts, 30 min before transformation, bottom plate was prepared
by pour 10ml Regeneration Agar medium containing 2× appropriate antibiotics into the
plate. Then, a second layer of Regeneration Agar medium (10ml, no antibiotics) was
poured onto the bottom plate before transformation. Afterwards, one aliquot of protoplast
was thawed on ice, 1 μl heparin (1 mg/ml) and 1-10 μl of DNA (3-5 μg) was added to the
protoplast and the mixture was incubated on ice for 10 min. 500 μl STC/PEG were then
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80
added to the protoplasts, mixed gently, and incubated for another 15 min on ice. Finally, the
transformation mixture was plated on regeneration agar plates. Transformants appeared
after 4-7 days were singled out and grown on PD-agar plates containing no antibiotics.
Single colonies were picked and correct transformants were determined by southern
blotting.
SCS 20 mM Na-citrate, pH 5.8
1 M Sorbitol
Add dH2O and autoclave
STC 10 mM Tris-Cl, pH 7.5
100 mM CaCl2
1 M Sorbitol
Add dH2O and autoclave
STC/PEG 40 % (w/v) PEG (MW: 3350)
Add STC and autoclave
4.6.2.2 Pathogenicity assays
Pathogenicity assays were performed as described by Kämper (2006). For maize (Zea
mays) infections, pre-cultures of U. maydis strains were grown in YEPSlight medium over
night. The culture was diluted in 50 ml YEPSlight medium to an OD600 of 0.4 and grew for
about 1 hour 45 min to an OD600 ≈ 1.0. The cells were then harvested and resuspended in
distilled water to an OD600 of 1.0 and injected into 7-day-old seedlings of the Early
Golden Bantam (Olds Seeds, Madison, WI). Plants were kept in the greenhouse with a
light-dark cycle of 16 (28 °C) and 8 hrs (20 °C). Disease symptoms were scored according
to severity 12 days after infection (Kämper et al., 2006).
4.6.3 S. cerevisiae methods
Liquid cultures were incubated at 28 °C at 200 rpm. Solid media were incubated under
aerobic condition at 28 °C. To prepare frozen stocks, exponentially growing cultures were
mixed with NSY-glycerol at a 1:1 ratio and stored at -80 °C.
4.6.3.1 Preparation of compentent cells and transformation of S. cerevisiae
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An overnight pre-culture of S. cerevisiae AH109 was diluted in 50ml YEPD medium at a
ratio 1:50 and grew until OD600 ≈ 0.5-0.7 (4-6 hours). The cells were harvested by
centrifuging at 2000 rpm/500 g, 3 min and room temperature. The pellet was then wash
once by 15 ml sterile H2O and once by 10 ml SORB. The cells were re-suspended in 400 µl
SORB supplemented with 40 µl carrier DNA (10 mg/ml Herring sperm DNA, denatured at
100 °C for 10 min and cooled on ice, Invitrogen). 50 μl of cell suspension were aliquoted
into pre-chilled 1.5 ml microcentrifuge tubes for immediate use, or stored at -80 °C for later
use.
For yeast transformation, the protocol of Burke was followed (Burke et al., 2000). An
aliquot of competent cells was thawed. Subsequently, plasmid DNA (maximal 2µl plasmid
DNA/10 µl competent cells) was added, gently mixed, then, 6 fold volume of the sterile
PEG was added gently mixed and incubated at 30 °C for 30 min. The mixture was then heat
shocked at 42 °C for 15 min and centrifuged at 2000 rpm, 3 min and room temperature. The
cell pellet was then washed once with 1ml YEPD medium. For the recovery of the S.
cerevisiae cells, the pellet was re-suspended in 1 ml YEPD medium and incubated at 500
rpm for 2-3 hours at 30 °C. Finally, the entire cell suspension was plated on SD medium
plate.
SORB 10 mM Tris-HCl, pH 8.0
100 mM lithium acetate
1 M sorbitol
1 mM EDTA
Add dH2O and autoclave
PEG 10 mM Tris-HCl, pH 8.0
100 mM lithium acetate
40 % PEG 3350
1 mM EDTA
Add dH2O and autoclave
4.6.3.2 Yeast two-hybrid assay
The yeast two hybrid analysis was performed using the matchmarker GAL4 two hybrid
system 3 (Clontech) following the manufacturer’s instructions. Transformants were
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82
spreaded on synthetic dropout medium plates without leucine and tryptophan. Growth
assays were tested on synthetic dropout medium plates either without leucine, tryptophan or
without Adenine, leucine, tryptophan and histidine.
4.6.3.3 Re-transformation and growth assay
The plasmids constructed on the backbone of either pGAD or pGBK were co-transformed
into AH109. The transformants were grown in SD-Leu-Trp medium overnight. The
cultures were adjusted to OD600≈ 1.0 and serial dilutions were spotted on the plates
containing low stringency medium (-Leu-Trp) and the plates containing high stringency
medium (-Leu-Trp-His-Ade) at the same time. The plates were incubated for three days
before observation. The expression of genes was determined by Western blotting using HA
antibody for activation domain and c-Myc antibody for DNA binding domain.
4.7 Molecular biological methods
Standard molecular biology methods are performed following protocols as described by
Ausubel and Sambrook (Ausubel et al., 1987, Sambrook et al., 1989). The concentration of
nucleic acids was determined by photometry (NanoDrop ND-1000 Spectrophotometer).
The plasmid DNA purification was performed using QIAprep spin miniprep kit (QIAGEN)
following the manufacturer’s instructions. DNA fragment was purified using Wizard® SV
Gel and PCR Clean-Up System (Promega) following the manufacturer’ instructions. The
preparation of genomic DNA from U. maydis was performed following the protocol of
Hoffman and Winston (Hoffman & Winston, 1987).
4.7.1 Southern blotting
10 µl of genomic DNA was digested overnight with respective restriction enzymes
(NdeI/BamHI for cbx locus) in 20 µl volume. Digestions were separated on a 1× TAE 0.8
% agarose gel for about 4 hours at 90 V. The gels were soaked in 0.25 M HCl solution with
shaking for 20-30 min until bromothymol blue turns yellow. HCl solution was then
replaced by 0.4 M NaOH and incubated for 20-30 min with shaking until the color turns
blue again. Subsequently, DNA was transferred from the gel to nylon membrane in 0.4 M
NaOH for 2 hours (change the tissue every 30 min). The membrane was UV cross-linked at
1200 µJoules (100×) (UV Stratalinker 1800, Stratagene). Dig-labeling probe was generated
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83
as described in the PCR DIG Labeling Mix protocol (Roche, Mannhein). The hybridization,
wash and exposure steps were performed following the protocol of (Sambrook et al., 1989).
4.7.2 Western blotting
Proteins were separated by SDS-PAGE and transferred to a PVDF (polyvinylidene
difluoride) membrane at 40-45 mA and 25 V for 1-2 hours. Afterwards, the membrane was
blocked with TBST buffer containing 5 % non-fat dry milk at room temperature for at least
one hour. The membrane was then incubated with respective primary antibody (Table. 15)
diluted in TBST buffer containing 3 % non-fat dry milk for at least one hour. Subsequently,
the membrane was washed three times with TBST buffer for 10 min. Then, the membrane
was incubated with respective secondary antibody (Table. 15) diluted in TBST buffer
containing 3 % non-fat dry milk for at least 1 hour. Subsequently, the membrane was
washed three times with TBST buffer for 10 min. Chemiluminescent detection was
performed using an ECL kit (Amersham Biosciences, cat. no. RPN-2106) following the
manufacturer’s instructions.
Antibody Source Suppler Working solution
α-c-myc mouse Sigma M5546 1:3,000
α -HA mouse Sigma H9658 1:3,000
α -BD mouse Santa Crutz biotechnology sc-510 1:2,000
α -AD mouse Santa Crutz biotechnology sc1663 1:2,000
Goat anti-Mouse IgG, Cy3 conjugate Goat Millipore AP124C 1: 300
Goat anti-Rabbit IgG, Cy3 conjugate Goat Millipore AP132C 1: 300
Rabbit anti-Mouse IgG Rabbit Life Technologies 61-6000 1: 500
α -mouse IgG, HRP-linked antibody horse Cell Signaling Technology #7076 1:10,000
TBST 50mM Tris-HCl, pH 7.4
150mM NaCl
0.1 % Tween-20
Add dH2O and autoclave
Table 15. Antibodies used in this study
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4.7.3 Isolation of Plasmid DNA from S. cerevisiae
The yeast cells were collected by centrifugation. Then, 0.4 ml ustilago-lysis buffer, a spoon
of glass beads and 0.4 ml phenol-chloroform was added subsequently into the tube. The
mixture was vibrated for 1,500 rpm for 15-30 min and centrifuged at 13,000 rpm for 5 min.
The upper layer was then transferred into a new 1.5 ml tube and 0.4 ml of chloroform was
added. The mixture was vibrated for 10 min and centrifuged at 13,000 rpm for 5 min.
Afterwards, the upper layer was transferred into a new 1.5 ml tube and 2.5×volume of 100
% ethanol was added into the new tube and mixed. After centrifugation, the pellet was
washed once by 70 % ethanol and incubated at room temperature for 10 min for drying.
The DNA was re-suspended in 30 µl of H2O.
Ustilago-lysis 50 mM Tris-HCl, pH 7.5
50 mM Na2-EDTA
1 % (w/v) SDS
Add dH2O
4.7.4 Protein extraction from S. cerevisiae
The yeast cells were harvested by centrifugation. Then, SDS-PAGE loading buffer
supplemented with 100mM DTT was added and the mixture was incubated at 100 °C for 10
min. Then, the mixture was vortexed for 5 min. This was followed by incubation at 100 °C
for 10 min again. Afterwards, the mixture was centrifuged for 5 min and the supernatant
was transfered into a new tube for loading onto SDS-PAGE.
4×SDS-PAGE Loading 150 mM Tris-HCl, pH 7.0
12 % SDS (w/v),
6 % β-mercaptoethanol (v/v)
0.05 % Coomassie blue G-250
30 % glycerol (w/v)
Add dH2O
4.8 Biochemical methods
The concentration of proteins was determined using Roti®-Quant (Proteinbestimmung nach
Bradford, Carl Roth) (Bradford, M., 1976, Anal. Biochem. 72, 248-254.) following the
Materials and methods
85
manufaturer’s instruction. Proteins was detected using SDS-PAGE Tris-Glycine gel system
or Tricine gel system described by Hermann Schägger depending on the size of the proteins
(Schagger, 2006).
4.8.1 Purification of GST-tagged protein
E. coli cells expressing recombinant protein were harvested by centrifugation at 8000 rpm
for 8 min at 4 °C (could be stored at -20°C for later purification if necessary). The cells
were dispersed in pre-chilled lysis buffer (PBS) at a ratio of 1 g/2-5 ml lysis buffer. After
adding PMSF, the cells were sonicated (Duty cylcle 50 %, output 4, 0.5 min/ 1.5 min×6)
and centrifuged for 30 min at 4 °C twice at 17,000 rpm. The supernatant was pipetted onto
PBS equilibrated glutathione sepharoseTM 4 fast flow resin (GE Healthcare, Uppsala). The
tube was applied onto test tube rotator and incubated at 4 °C for 2 hours. The tube was
centrifuged at 500 g for 5min at 4 °C to remove the supernatant and washed three times. To
elute the protein from the resin, 1 CV (column volume) GST elution buffer was added and
incubated for 20 min at room temperature. This step was repeat twice and the three elutes
were pooled together.
100×PMSF stock solution (10 mM) 1.74 mg/ml PMSF
Add isopropanol and store at -20 °C
10×PBS 17.8 g Na2HPO4. 2H2O pH 7.4
2.4 g KHPO4
2 g KCl
80 g NaCl
Add dH2O to 1L and autoclave
GST elution buffer 20 mM glutathione
Add PBS and sterile filtered
4.8.2 Purification of Strep-tagged protein
E. coli cells were harvested and dispersed in pre-chilled buffer W at a ratio of 1 g/2-5 ml
lysis buffer. Lysozyme, DNase I and Protease Inhibitor Cocktail (Roche Diagnostics) was
added to the suspension and incubated for 30 min. The cells were broken using French
Materials and methods
86
pressure cell press (SLM Aminco) at high ratio, 1000 psi. The lysate was centrifuged for 30
min at 4 °C twice at 17,000 rpm. The supernatant was then pipetted onto buffer W
equilibrated column filled with Strep-Tactin Resins (IBA). The column was washed five
times with buffer W. Finally, the protein was eluted by 6×0.5 CV buffer E. The resin was
regenerated by wash with 3× 5 CV buffer R.
Buffer W (washing buffer) 100 mM Tris-HCl pH 8.0 150 mM NaCl 1 mM EDTA Add H2O and autoclave Buffer W (washing buffer) 100 mM Tris-HCl pH 8.0
150 mM NaCl
1 mM EDTA
2.5 mM desthiobiotin
Add H2O and autoclave
Buffer W (washing buffer) 100 mM Tris-HCl pH 8.0
150 mM NaCl
1 mM EDTA
1 mM HABA (hydroxy-azophenyl-
benzoic acid)
Add H2O and autoclave
4.8.3 Purification of His-tagged protein
The cell lysate was prepared as describe in strep-tagged protein purification above. The
supernatant was then pipetted onto his lysis buffer equilibrated column filled with Ni-NTA-
agarose (Qiagen). The column was washed twice with his lysis buffer. Finally, the protein
was eluted by 4 × 1.5 CV his elution buffer.
The protein purified from NTA-agarose was diluted 3-5 times and loaded onto MonoQ TM
5/50 GL column in AKTA FPLC System (GE Healthcare). After washing by 2 CV MonoQ
start buffer, the protein was eluted by gradient elution with 25 CV from 0 % MonoQ
elution buffer to 60 % MonoQ elution buffer. The column was cleaned by 5 CV MonoQ
elution buffer and equilibrated by 5 CV MonoQ start buffer.
Materials and methods
87
The protein purified from MonoQ column was concentrated on Amicon Ultra-4 3 kDa
columns (Millipore) at 3000 g. The concentrated protein was loaded onto the SuperdexTM
75 10/300 GL (GE Healthcare) column and eluted using Gel filtration buffer at fraction size
0.5 ml and flow rate 0.5 ml/min.
His lysis 20 mM Tris-HCl pH 7.4
150 mM NaCl
20 mM Imidazole
1 mM EDTA
Add H2O and sterile filtered
His elution 20 mM Tris-HCl pH 7.4
150 mM NaCl
0.5 M Imidazole
1 mM EDTA
Add H2O and sterile filtered
MonoQ start 20 mM Tris-HCl pH 7.0
1 mM EDTA
Add H2O and sterile filtered
MonoQ elution 20 mM Tris-HCl pH 7.0
1 M NaCl
1 mM EDTA
Add H2O and sterile filtered
Gel filtration 15 mM Tris-HCl pH 7.0
5 mM NaCl
Add H2O and sterile filtered
4.8.4 Protein purification from N. benthamiana
Frozen tissue (about 4 leaves) was ground in liquid nitrogen, re-suspended in 6 ml BP
buffer and centrifuged at 40,000g for 10 min at 4 °C. The supernatant was filtered through
a filter pipette into a 15 ml tube with the IgG beads (Mouse IgG-Agarose, A0919, Sigma)
Materials and methods
88
equilibrated with BP buffer without protease inhibitors. The mixture was incubate on a test
tube rotator for 2 hours at 4 °C and centrifuged for 5 min at 1500 g. The supernatant was
removed and 500 µl BP buffer was added into the tube and transferred into a 1.5 ml tube.
The beads were then washed by 0.5 ml BP once and 1 ml BP2 twice. 50 µl BP2 containing
the cleavage protease was added into the tube and incubated on a test tube rotator for at
least 16 hours. Finally, the beads were span down and the supernatant containing the
protein was transferred into a new tube.
BP 100 mM Tris-HCl pH 7.5
100 mM NaCl
5 mM EDTA
5 mM EGTA
10 mM NaF
0.1 % Triton-X100
1 mM β-mercaptoethanol
10 % Glycerol
0.1 mM NaVO3
10 mM β-GPh
Protease inhibitors
Add H2O and sterile filtered
BP2 50 mM HEPES pH 7.0
150 mM NaCl
1 mM DTT
0.1 % Triton-X100
Add H2O and sterile filtered
1000×Protease inhibitors 1 M Benzamidine in ethanol
10 mg/ml Aprotinin in H2O
1 M 1-10 phenantroline in ethanol
Materials and methods
89
4.8.5 Cysteine pretease activity and inhibition assay
The cyteine protease was activated by adding 10 mM DTT, adjusting pH to pH 4.0-4.5 with
acetic acid and incubating at 30 °C for 1 hour. The enzymatic activity was tested in a 200 µl
volume containing 100 µl NaPi buffer, cysteine protease, and 2 µM substrate (Z-Phe-Arg-
AMC, PeptaNova). The progress of the reaction was monitored using fluorimeter
(SAFIRE, Tecan).
NaPi Buffer 0.1 M Na phosphate pH 6.0
Add H2O and autoclave
4.9 Staining and microscopy observation
All the confocal images were taken using a TCS-SP5 confocal microscopy (Leica). WGA-
AF488 was excited was at 488 nm and detected at 500-540 nm; Propidium Iodide was
excited was at 561 nm and detected at 580-660 nm; Anniline blue was excited was at 405
nm and detected at 490-520 nm; DAPI was excited was at 405 nm and detected at 430-480
nm; Cy3 was excited was at 561 nm and detected at 573-610 nm; mCherry was excited was
at 561 nm and detected at 573-610 nm.
4.9.1 WGA-AF488 / Propidium Iodide staining
Infected leaf tissue was harvested and cleared in ethanol overnight (or longer). The samples
were then treated with 10 % KOH at 85 °C for 3-4 h. After washing with PBS for 2-4
times, the samples were incubated in staining solution for 30 min (vacuum infiltrate the
samples three times for about 1-2 min) and destained in 1x PBS. Then, the samples were
stored in dark at 4 °C until analysis. By this stain, fungal hyphae were stained with
Fluorescein WGA (Vector Laboratories); Plant membranes were visualized using
Propidium Iodide (Fluka).
Staining solution 20 µg/ml Propidium Iodide
10 µg/ml WGA-AF
0.02 % Tween-20
PBS (pH 7.4)
Materials and methods
90
4.9.2 Anniline blue / Propidium Iodide staining
Infected leaf tissue was harvested and rinsed twice with 50 % ethanol. The samples were
then rinsed with 0.1 M Na2HPO4 buffer (pH≈9.0 without adjusting). Afterwards, incubate
the samples in 20 µg/ml Propidium Iodide for 30 min and rinse the samples with 0.1 M
Na2HPO4 buffer again. Subsequently, freshly prepared 0.05 % aniline blue (w/v in 0.1 M
Na2HPO4 buffer) was added and incubated for 1 hour in dark. The sample was finally
prepared on slides in 0.05 % aniline blue.
4.9.3 Chlorazol Black E staining
Infected leaf tissue was harvested and cleared in ethanol overnight (or longer). The samples
were then treated with 10 % KOH at 85 °C for 3-4 h. After washing with dH2O once, the
samples was soak in chlorazol Black E staining solution (0.03 % Chlorazol black E in a
1:1:1 solution of dH2O, lactic acid and glycerol) overnight at 60 °C. Afterwards, the
samples were destained (or stored) for 3 days in 50 % glycerol.
91
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Supplementary data
The data CD contains the following files:
Supplementary Table. 1, Differentially expressed maize genes.xlsx
Supplementary Table. 2, Enriched biological processes.xlsx
Supplementary Table. 3, Early defense response genes.xlsx
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Acknowledgements
With great pleasure, I would like to express my sincere gratitude to all who contribute to
this thesis.
First and foremost I thank my thesis supervisor, Prof. Dr. Regine Kahmann for offering me
such a challenging and promising project. A warm reception in a snowing day on 2009
started her three and a half years’ encouragement, trust, guidance and thoughtful support.
This enable me to overcome all the hardness and frustrations and learn not only science
itself but also the dedication, insistence, and important of precise and scientific ethics. In
particular, I would like to thank her insightful guidance during my writing. I am deeply
greetful to my advisory and thesis committee members, Prof. Dr. Michael Bölker, Prof. Dr.
Martin Thanbichler, Prof. Dr. Alfred Batschauer and Dr. Seigo Shima for their time and
constructive suggestions.
My deep and sincere thanks go to current and former colleagues from Kahmann lab and
department of orgnismic interactions for the precious suggestions, encouragement and help.
Special thanks to Dr. Armin Djamei, Dr. Stefanie Reißmann, Dr. Julien Yann Dutheil, Dr.
Kerstin Schipper, Dr. Christian Herrberger for their help in data process and interpretation,
discussion, encouragement and valuable suggestions. I am sincerely greatful to Karin
Münch, Nicole Rössel, Volker Vincon for encouragement and assistance in the lab, Rolf
Rösser for computational support, Stefan Schmidt, Anita Boos, Ria Faber, Vera
Matschiske-Peters, Claudia Schäfer for their assitance and generous help. I thank our
collaborators Prof. Dr. Michael Groll (TUM), Prof. L. Søgaard-Andersen (MPI) and Dr.
Bruno Huettel (MPGC) and IMPRS-Mic Marburg for all the support to promote the
development of this project. I would like to extend my thanks to Mr. Christian
Bengelsdorff, Susanne Rommel and Simone Hain for taking care of all the documents. I
would like to thank the financial support through SFB593 and Synmikro.
Finally, I am indebted to my family. I would like to express my heatily thankful to my
parents, Liang Yingchun and Zhou Ping and my parents-in-law, Wang Mingliang and Zeng
Xianping for their everlasting love and support; my wife, Li Fujin for her love,
encouragement and care of my life and my son, Liang Yiheng who was born during my
writing and provided us with plenty of joys and moral support.
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Curriculum Vitae
Personal information
Name Liang Liang
Date of birth Dec 1979
Place of birth Hebei, P. R. China
Education
Jan 2009-Jun 2012 Philipps-Universität Marburg
Max-Planck-Institute for Terrestrial Microbiology
Marburg/Lahn, Germany
PhD thesis: The role of Stp1, a secreted effector, in the
biotrophic interaction of Ustilago maydis and its host plant
maize
Jul 2005–Jan 2008 Institute of Microbiology, Chinese Academy of Sciences
Beijing, P. R. China
Master of Science. Master thesis: The function of iunH and
gerA in spore germination of Bacillus thuringiensis
Sep. 2004-Jul. 2005 Graduate school of Chinese Academy of Sciences
Beijing, P. R. China
Sep. 2000-Jun. 2004 Hebei University
Baoding, P. R. China.
Bachelor of Science